Polyhydroxy alkanoate copolymer including within molecule unit having vinyl group or carboxyl group in side chain, and producing method therefor

ABSTRACT

The invention provides a PHA copolymer including at least a 3-hydroxy-ω-carboxyalkanoic acid represented by a formula (19) or (32) and simultaneously at least a unit represented by a formula (2) or a formula (3) in a molecule, a precursor PHA copolymer having a corresponding vinyl group or a corresponding alkoxycarbonyl group, a biosynthesis method thereof by microorganisms, and a method of producing a desired PHA copolymer from the precursor PHA copolymer: (wherein k, m, n are integers; R 18  represents H, Na, K, R 27? represents (A); R 1  represents a substituent on a cyclohexyl group and represents H, CN, NO 2 , a halogen atom, CH 3 , C 2 H 5 , C 3 H 7 , CF 3 , C 2 F 5 , or C 3 F 7 ; R includes a residue including a phenyl structure or a thienyl structure; these being independent for each unit).

TECHNICAL FIELD

The present invention relates to a polyhydroxy alkanoate (hereinafteralso abbreviated as PHA) copolymer including a novel unit having adouble bond and a producing method therefor utilizing microorganisms,also polyhydroxy alkanoate copolymer including a novel unit having acarboxyl group or a salt thereof, derived from the aforementionedcopolymer, and a producing method therefor.

Also the present invention relates to a polyhydroxy alkanoate copolymerincluding a novel unit having an ester group and a producing methodtherefor utilizing microorganisms, also polyhydroxy alkanoate copolymerincluding a novel unit having a carboxyl group or a salt thereof,derived from the aforementioned copolymer, and a producing methodtherefor.

BACKGROUND ART

It has already been reported that various microorganisms producepoly-3-hydroxybutyric acid (PHB) or other poly-3-hydroxyalkanoate (PHA)and accumulate such products therein. Such PHA produced by themicroorganisms can be utilized for producing various products. Also thePHA produced by microorganisms, being biodegradable, has the advantagethat it can be completely decomposed by the microorganisms. Thereforethe PHA produced by microorganisms, when discarded, unlike the variousconventional synthesized polymers, would not cause pollution resultingfrom remaining in the natural environment. Also the PHA produced bymicroorganisms shows satisfactory affinity to the living tissues and isexpected in the applications as the soft material for medical use.

However, for wider application of microorganism-produced PHA, forexample for application as functional polymer, PHA having a substituentother than alkyl group in the side chain, namely “unusual PHA”, isanticipated to be extremely useful. Examples of hopeful substituents forthis purpose include a group containing an aromatic ring (phenyl group,phenoxy group etc.), an unsaturated hydrocarbon group, an ester group,an allyl group, a cyano group, a halogenated hydrocarbon group and anepoxide present on the side chain. Among these, PHA having an aromaticring is actively investigated as follows:

-   -   (a) PHA containing a phenyl group or a partially substituted        group thereof:

Makromol. Chem. 191, 1957-1965(1990) and Macromolecules, 24,5256-5260(1991) report that Pseudomonas oleovorans produces PHAcontaining 3-hydroxy-5-phenylvaleric acid as a unit, from5-phenylvaleric acid as a substrate.

Macromolecules, 29, 1762-1766(1996) reports that Pseudomonas oleovoransproduces PHA containing 3-hydroxy-5-(p-tolyl)valeric acid as a unit,from 5-(p-tolyl)valeric acid as a substrate.

Macromolecules, 32, 2889-2895(1999) reports that Pseudomonas oleovoransproduces PHA containing 3-hydroxy-5-(2,4-dinitrophenyl)valeric acid and3-hydroxy-5-(p-nitrophenyl)valeric acid as units, from5-(2,4-dinitrophenyl)valeric acid as a substrate.

-   -   (b) PHA containing phenoxy group or a partially substituted        group thereof:

Macromol. Chem. Phys., 195, 1665-1672(1994) reports that Pseudomonasoleovorans produces a PHA copolymer containing3-hydroxy-5-hydroxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid asthe units, from 11-phenoxyundecanoic acid as a substrate.

Also Japanese Patent No. 2989175 discloses inventions relating to ahomopolymer constituted of a 3-hydroxy-5-(monofluorophenoxy) pentanoate(3H5(MFP)P) unit or a 3-hydroxy-5-(difluorophenoxy) pentanoate(3H5(DFP)P) unit, a copolymer containing either a 3H5(MFP)P unit or a3H5(DFP)P unit or both, a novel strain of Pseudomonas putida capable ofproducing these polymers, and a method for producing the aforementionedpolymers utilizing bacteria of genus Pseudomonas. This patentspecification teaches, as the effects of such inventions, that PHApolymer having a phenoxy group substituted with 1 or 2 fluorine atoms atthe end of the side chain can be biosynthesized from a long-chain fattyacid having a fluorine substituent and that thus produced PHA has a highmelting point and is capable of providing stereoregularity and waterrepellency while maintaining satisfactory working properties.

In addition to the fluorine-substituted PHA having a fluorinesubstitution on the aromatic ring in the unit, there are alsoinvestigated PHA having a cyano group or a nitro group on the aromaticring in the unit.

Can. J. Microbiol., 41, 32-43(1995) and Polymer International, 39,205-213(1996) report production of PHA, containing3-hydroxy-6-(p-cyanophenoxy) hexanoic acid or3-hydroxy-6-(p-nitrophenoxy) hexanoic acid as the monomer unit, byPseudomonas oleovorans ATCC 29347 strain and Pseudomonas putida KT2442strain, from octanoic acid and 6-(p-cyanophenoxy) hexanoic acid or6-(p-nitrophenoxy) hexanoic acid as a substrate.

These references relate to PHA having an aromatic ring on the sidechain, instead of alkyl groups of the usual PHA, which are effective inobtaining polymer with physical properties resulting from such aromaticring.

Also as a new category not limited to changes in the physicalproperties, investigations are also made for producing PHA having anappropriate functional group on the side chain, thereby obtaining PHAwith new functions utilizing such substituent.

As a specific method for such purpose, investigations are also made forproducing PHA having, in a unit thereof, reactive group such as a bromogroup or a vinyl group with a high activity for example in an additionreaction to introduce an arbitrary function group in a side chain of thepolymer by a chemical conversion utilizing such active group, in orderto obtain PHA of multiple functions.

Macromol. Rapid Commun., 20, 91-94(1999) reports production of PHAhaving a bromo group in a side chain by Pseudomonas oleovorans, andmodifying the side chain with a thiolated product of acetylated maltosethereby synthesizing PHA different in solubility and hydrophilicity.

Polymer, 41, 1703-1709(2000) reports producing PHA, having3-hydroxyalkenic acid with an unsaturated bond (vinyl group) at an endof a side chain as a monomer unit, by Pseudomonas oleovorans with10-undecenoic acid as a substrate, followed by an oxidation reactionwith potassium permanganate to synthesize 3-hydroxyalkanoic acid havinga diol at the end of the side chain, which PHA is reported to show sucha change in solubility in solvents, as becoming soluble in polarsolvents such as methanol, an acetone-water (80/20, v/v) ordimethylsulfoxide and insoluble in non-polar solvents such aschloroform, tetrahydrofuran or acetone.

Also Macromolecules, 31, 1480-1486(1996) reports production of apolyester, including a unit having vinyl group in a side chain byPseudomonas oleovorans and epoxylating the vinyl group to obtain apolyester having an epoxy group in the side chain.

Also Polymer, 35, 2090-2097(1994) reports a crosslinking reaction withinthe polyester molecule utilizing the vinyl group in the side chain ofpolyester, thereby improving physical properties of polyester.

Macromolecular chemistry, 4, 289-293(2001) reports producing PHA,including 3-hydroxy-10-undecenoic acid as a monomer unit, from10-undecenoic acid as a substrate, and then executing an oxidationreaction with potassium permanganate to obtain PHA including3-hydroxy-10-carboxydecanoic acid as a monomer unit, and reports animprovement in a decomposition thereof.

Furthermore, in order to modify physical properties of PHA having anactive group in a unit and to actually utilize it as a polymer, it hasbeen studied biosynthesis of a PHA copolymer including a unit having theactive group and other units; Macromolecules, 25, 1852-1857(1992)reports production of a PHA copolymer including a3-hydroxy-ω-bromoalkanoic acid unit and a linear alkanoic acid unit byPseudomonas oleovorans in the presence of an ω-bromoalkanoic acid suchas 11-bromoundecanoic acid, 8-bromooctanoic acid or 6-bromohexanoic acidand n-nonanoic acid.

Such PHA having a highly reactive active group such as a bromo group ora vinyl group can be subjected to introduction of various functionalgroups or chemical modification, and such a group can be a crosslinkingpoint for a polymer, so that it is very useful means for realizingmultiple functions in PHA.

Also technologies related to the present invention include a technologyof oxidizing a carbon-carbon double bond with an oxidant to obtain acarboxylic acid (Japanese Patent Application Laid-Open No. S59-190945,J. Chem. Soc., Perkin. Trans. 1, 806(1973), Org. Synth., 4, 698(1963),J. Org. Chem., 46, 19(1981), and J. Am. Chem. Soc., 81, 4273(1959).

On the other hand, active investigations are being made for obtaining amulti-functional PHA from PHA including an ester group in a unit.

Macromol. Chem. Phys., 195, 1405-1421(1994) reports production of apolyhydroxy alkanoate including a unit having an ester group in a sidechain, employing Pseudomonas oleovorans as a production microorganismand an alkanoate ester.

Also University of Massachusetts Ph. D. Dissertation Order Number9132875 (1991) reports production of a polyhydroxy alkanoate including aunit having a benzylester structure, also employing Pseudomonasoleovorans as a production microorganism.

However, the copolymers in the foregoing reports are comprised of amonomer unit having a carboxyl group or an ester group at the end of aside chain and a monomer unit having a linear alkyl group (usual PHA)having a low glass transition temperature. On the other hand, there isno report on copolymers including unusual PHA having on the side chainthereof a substituent other than a linear alkyl group, such as a phenylstructure, a thienyl structure or a cyclohexyl structure. Thus. suchpolyhydroxy alkanoate and a producing method therefor have beenrequired.

Also PHA having a vinyl group as an active group is a PHA copolymer witha monomer unit having a linear alkyl group(usual PHA), its low glasstransition temperature and low melting point are undesirable propertiesin the working and the use of the polymer.

Because of the above-described situation, there have been a demand forPHA having an active group and a production method therefor, such thatPHA can be produced by a microorganism at a high yield, the unit ratioof the active group can be controlled, and its physical properties canbe freely regulated not to limit its application as a polymer.

DISCLOSURE OF THE INVENTION

As a result of intensive investigations, the present inventors havefound a method of synthesizing a PHA formed by copolymerization of aunit having a vinyl group, an ester group or a carboxyl group of a highreactivity, and a unit having either one of a phenyl structure, athienyl structure and a cyclohexyl structure which can contribute to animprovement of physical properties of the polymer, and have thus madethe present invention.

The present invention is outlined in the following.

-   [1] A polyhydroxy alkanoate copolymer including at least a    3-hydroxy-ω-alkenoic acid unit represented by a chemical formula (1)    in a molecule, and simultaneously at least a 3-hydroxy-ω-alkanoic    acid unit represented by a chemical formula (2) or a    3-hydroxy-ω-cyclohexylalkanoic acid unit represented by a chemical    formula (3) in the molecule:    [Chemical Formula (1)]

in which n represents an integer selected within a range indicated inthe chemical formula; and in case plural units are present, n is thesame or different for each unit;[Chemical Formula (2)]

in which m represents an integer selected within a range indicated inthe chemical formula; R represents a residue having any of a phenylstructure or a thienyl structure; and in case plural units are present,m and R are the same or different for each unit;[Chemical Formula (3)]

in which R₁ being a substituent on a cyclohexyl group represents ahydrogen atom, a CN group, a NO₂ group, a halogen atom, a CH₃ group, aC₂H₅ group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F₇ group; krepresents an integer selected within a range indicated in the chemicalformula; and in case plural units are present, R₁ and k may be the sameor different for each unit.

-   [2] A polyhydroxy alkanoate copolymer including at least a    3-hydroxy-ω-carboxyalkanoic acid unit represented by a chemical    formula (19) or 3-hydroxy-ω-alkoxycarbonylalkanoic acid unit    represented by a chemical formula (32) in a molecule, and    simultaneously at least a 3-hydroxy-ω-alkanoic acid unit represented    by the chemical formula (2) or a 3-hydroxy-ω-cyclohexylalkanoic acid    unit represented by the chemical formula (3) in the molecule,    [Chemical Formula (19)]

in which n represents an integer selected within a range indicated inthe chemical formula; R₁₈ represents an H atom, a Na atom or a K atom:and in case plural units are present, n and R₁₈ may be the same ordifferent for each unit; and[Chemical Formula (32)]

in which n represents an integer selected within a range indicated inthe chemical formula; R₂₇ represents any of residues indicated in thechemical formula; and in case plural units are present, n and R₂₇ may bethe same or different for each unit.

-   [3] A method for producing a polyhydroxy alkanoate copolymer    including at least a 3-hydroxy-ω-alkenoic acid unit represented by    the chemical formula (1) in a molecule, and simultaneously at least    a 3-hydroxy-ω-alkanoic acid unit represented by a chemical    formula (2) or a 3-hydroxy-ω-cyclohexylalkanoic acid unit    represented by a chemical formula (3) in the molecule, characterized    in including a biosynthesis by a production microorganism from at    least an ω-alkenoic acid represented by a chemical formula (24) and    at least a compound represented by a chemical formula (25) or at    least an ω-cyclohexylalkanoic acid represented by a chemical    formula (26) as starting materials:    [Chemical Formula (24)]

in which p represents an integer selected within a range indicated inthe chemical formula;[Chemical Formula (25)]

in which q represents an integer selected within a range indicated inthe chemical formula; and R₂₃ includes a residue having a phenylstructure or a thienyl structure;[Chemical Formula (26)]

in which R₂₄ represents a substituent on a cyclohexyl group andrepresents an H atom, a CN group, a NO₂ group, a halogen atom, a CH₃group, a C₂H₅ group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F₇group; and r represents an integer selected within a range indicated inthe chemical formula.

-   [4] A method for producing a polyhydroxy alkanoate copolymer    including at least a 3-hydroxy-ω-carboxyalkanoic acid unit    represented by the chemical formula (19) in a molecule, and    simultaneously at least a 3-hydroxy-ω-alkanoic acid unit represented    by the chemical formula (2) or a 3-hydroxy-ω-cyclohexylalkanoic acid    unit represented by the chemical formula (3) in the molecule    comprising the steps of:

preparing a polyhydroxy alkanoate copolymer including at least a3-hydroxy-ω-alkenoic acid unit represented by the chemical formula (1)in a molecule, and simultaneously at least a 3-hydroxy-ω-alkanoic acidunit represented by the chemical formula (2) or a3-hydroxy-ω-cyclohexylalkanoic acid unit represented by the chemicalformula (3) in the molecule as a starting material, and oxidizing adouble bond portion in the polyhydroxy alkanoate represented in thechemical formula (1) to generate the object polyhydroxy alkanoatecopolymer.

-   [5] A method for producing a polyhydroxy alkanoate copolymer,    characterized in employing a polyhydroxy alkanoate copolymer    including at least a 3-hydroxy-ω-alkoxycarbonylalkanoic acid unit    represented by a chemical formula (32) in a molecule, and    simultaneously at least a 3-hydroxy-ω-alkanoic acid unit represented    by the chemical formula (2) or a 3-hydroxy-ω-cyclohexylalkanoic acid    unit represented by the chemical formula (3) in the molecule as a    starting material, and executing a hydrolysis in the presence of an    acid or an alkali or executing a hydrogenolysis including a    catalytic reduction, thereby generating a polyhydroxy alkanoate    copolymer including at least a 3-hydroxy-ω-carboxyalkanoic acid unit    represented by the chemical formula (19) in a molecule, and    simultaneously at least a 3-hydroxy-ω-alkanoic acid unit represented    by the chemical formula (2) or a 3-hydroxy-ω-cyclohexylalkanoic acid    unit represented by the chemical formula (3) in the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹H-NMR spectrum of a polyester obtained in Example 1.

FIG. 2 is a ¹H-NMR spectrum of a polyester obtained in Example 2.

FIG. 3 is a ¹H-NMR spectrum of a polyhydroxy alkanoate copolymerobtained in Example 11, and including3-hydroxy-5-(phenylsulfanyl)valeric acid represented by a chemicalformula (58), a 3-hydroxy-10-undecenoic acid represented by a chemicalformula (5), 3-hydroxy-8-noneic acid represented by a chemical formula(6) and 3-hydroxy-6-heptenic acid represented by a chemical formula (7).

FIG. 4 is a ¹H-NMR spectrum of a polyhydroxy alkanoate copolymerobtained in Example 11, and including3-hydroxy-5-(phenylsulfonyl)valeric acid represented by a chemicalformula (59), a 3-hydroxy-9-carboxynonanoic acid represented by achemical formula (54), 3-hydroxy-7-carboxyheptanoic acid represented bya chemical formula (55) and 3-hydroxy-5-carboxyvaleric acid representedby a chemical formula (56).

FIG. 5 is a GC-MS TIC spectrum of a methylation decomposite of apolyester obtained in Example 34.

FIG. 6 is a mass spectrum of a peak derived from a unit shown by achemical formula (80) of a methylation decomposite of the polyester,obtained in Example 34.

FIG. 7 is a mass spectrum of a peak derived from a unit shown by achemical formula (81) of a methylation decomposite of the polyesterobtained in Example 34.

FIG. 8 is a mass spectrum of a peak derived from a unit shown by achemical formula (82) of a methylation decomposite of the polyesterobtained in Example 34.

BEST MODE FOR CARRYING OUT THE INVENTION

A polyhydroxy alkanoate copolymer, the final product of the presentinvention, is a polyhydroxy alkanoate copolymer (hereinafter also calledcarboxyl PHA) comprising a unit having a carboxyl group on a side chainas represented by a chemical formula (19) and a unit represented by achemical formula (2) or a chemical formula (3):

in which n represents an integer selected within a range indicated inthe chemical formula; R₁₈ represents an H atom, a Na atom or a K atom;

in which m represents an integer selected within a range indicated inthe chemical formula; R includes a residue having any of a phenylstructure or a thienyl structure; and in case plural units are present,m and R may be the same or different for each unit;

in which R₁ represents a substituent on a cyclohexyl group andrepresents a hydrogen atom, a CN group, a NO₂ group, a halogen atom, aCH₃ group, a C₂H₅ group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or aC₃F₇ group; k represents an integer selected within a range indicated inthe chemical formula; and in case plural units are present, R₁ and k maybe the same or different for each unit.

In the present invention, R in the chemical formula (2) represents aresidue having a phenyl structure or a thienyl structure selected fromthe group consisting of chemical formulas (8), (9), (10), (11), (12),(13), (14), (15), (16), (17) and (18):

the chemical formula (8):

represents a group of non-substituted or substituted phenyl groups inwhich R₂, a substituent on an aromatic ring and represents an H atom,represents a halogen atom, a CN group, a NO₂ group, a CH₃ group, a C₂H₅group, a C₃H₇ group, a CH═CH₂ group, a COOR₃ group (R₃ represents an Hatom, a Na atom or a K atom) which is not included when produced by amicroorganism, a CF₃ group, a C₂F₅ group, or a C₃F₇ group; and in caseplural units are present, R₂ is the same or different for each unit;

the chemical formula (9):

represents a group of non-substituted or substituted phenoxy groups inwhich R₄ represents a substituent on an aromatic ring and represents anH atom, a halogen atom, a CN group, a NO₂ group, a CH₃ group, a C₂H₅group, a C₃H₇ group, a SCH₃ group, a CF₃ group, a C₂F₅ group, or a C₃F₇group; and in case plural units are present, R₄ may be the same ordifferent for each unit;

the chemical formula (10):

represents a group of non-substituted or substituted benzoyl groups inwhich R₅ represents a substituent on an aromatic ring and represents anH atom, a halogen atom, a CN group, a NO₂ group, a CH₃ group, a C₂H₅group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F₇ group; and incase plural units are present, R₅ may be the same or different for eachunit;

the chemical formula (11)

represents a group of substituted or non-substituted phenylsulfanylgroups in which R₆ represents a substituent on an aromatic ring andrepresents an H atom, a halogen atom, a CN group, a NO₂ group, a COOR₇group, a SO₂R₈ group (R₇ represents either one of H, Na, K, CH₃ andC₂H₅; and R₈ represents either one of OH, ONa, OK, a halogen atom, OCH₃and OC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group, a (CH₃)₂—CH groupor a (CH₃)₃—C group; and in case plural units are present, R₆ may be thesame or different for each unit;

the chemical formula (12):

represents a group of substituted or non-substituted(phenylmethyl)sulfanyl groups in which R₉ represents a substituent on anaromatic ring and represents an H atom, a halogen atom, a CN group, aNO₂ group, a COOR₁₀ group, a SO₂R₁₁ group (R₁₀ represents either one ofH, Na, K, CH₃ and C₂H₅; and R₁₁ represents either one of OH, ONa, OK, ahalogen atom, OCH₃ and OC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group,a (CH₃)₂—CH group or a (CH₃)₃—C group; and in case plural units arepresent, R₉ may be the same or different for each unit;

the chemical formula (13):

represents a 2-thienyl group;

the chemical formula (14)

represents a 2-thienylsulfanyl group;

the chemical formula (15):

represents a 2-thienylcarbonyl group;

the chemical formula (16):

represents a group of substituted or non-substituted phenylsulfinylgroups in which R₁₂ represents a substituent on an aromatic ring andrepresents an H atom, a halogen atom, a CN group, a NO₂ group, a COOR₁₃group, a SO₂R₁₄ group (R₁₃ represents either one of H, Na, K, CH₃ andC₂H₅; and R₁₄ represents either one of OH, ONa, OK, a halogen atom, OCH₃and OC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group, a (CH₃)₂—CH groupor a (CH₃)₃—C group; and in case plural units are present, R₁₂ may bethe same or different for each unit;

the chemical formula (17):

represents a group of substituted or non-substituted phenylsulfonylgroups in which R₁₅ represents a substituent on an aromatic ring andrepresents an H atom, a halogen atom, a CN group, a NO₂ group, a COOR₁₆group, a SO₂R₁₇ group (R₁₆ represents either one of H, Na, K, CH₃ andC₂H₅; and R₁₇ represents either one of OH, ONa, OK, a halogen atom, OCH₃and OC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group, a (CH₃)₂—CH groupor a (CH₃)₃—C group; and in case plural units are present, R₁₅ may bethe same or different for each unit; and

the chemical formula (18):

represents a (phenylmethyl)oxy group.

The producing methods therefor are mainly classified to:

a method of oxidizing a double bond portion in a polyhydroxy alkanoatecopolymer (hereinafter also called a precursor vinyl PHA) including a3-hydroxy-ω-alkenoic acid unit having a carbon-carbon double bond at anend of a side chain as represented in a chemical formula (1)

in which n represents an integer selected within a range indicated inthe chemical formula; and in case plural units are present, such unitsmay be mutually different), and a unit represented by a chemical formula(2) or a chemical formula (3); and

a method of hydrolyzing an alkoxycarbonyl portion in a polyhydroxyalkanoate copolymer (hereinafter also called an alkoxycarbonyl PHA)including a 3-hydroxy-ω-alkoxyalkanoic acid unit having an ester groupat an end of a side chain as represented in a chemical formula (48):

in which n represents an integer selected within a range indicated inthe chemical formula; R₄₇ represents any of residues indicated in thechemical formula; and in case plural units are present, n and R₄₁ may bethe same or different for each unit, and a unit represented by achemical formula (2) or a chemical formula (3). In the following, theprecursor vinyl PHA and the precursor alkoxycarbonyl PHA may becollectively called a precursor PHA.

A producing method for such precursor PHA is not particularlyrestricted, but there can be employed a microbial production usingmicroorganisms, a method using a genetically modified plant, or achemical polymerization. Preferably a method by microbial production isemployed.

The precursor vinyl PHA and the precursor ester (alkoxycarbonyl) PHAwere synthesized for the first time by the present inventors, and thepresent invention therefore includes also the precursor vinyl PHA andthe precursor ester PHA themselves, and a production process thereof bymicroorganisms. Also such precursor vinyl PHA and precursor ester PHAcan be effectively utilized not only for the carboxyl PHA which is anobject of the present invention but also for introducing otherfunctional groups.

In the following, there will be explained a producing method employingeach precursor PHA.

The precursor vinyl PHA can be producing by culturing a microorganism ina culture medium including an ω-alkenoic acid represented by a chemicalformula (24) and a compound represented by a chemical formula (25) or anω-cyclohexylalkanoic acid represented by a chemical formula (26).

Similarly, the precursor alkoxycarbonyl PHA can be producing byculturing a microorganism in a culture medium including a carboxylicacid monoester compound represented by a chemical formula (49) and acompound represented by the chemical formula (25) or anω-cyclohexylalkanoic acid represented by the chemical formula (26).

The chemical formulas (24), (49), (25) and (26) are as follows:

Chemical Formula (24)

Chemical Formula (49)

(wherein p is an integer selected within a range indicated in thechemical formula; and R₄₈ is either one of residues shown in thechemical formula.)Chemical Formula (25)

(wherein q is an integer selected within a range indicated in thechemical formula; and R₂₃ represents a residue including a phenylstructure or a thienyl structure.)Chemical Formula (26)

(wherein R₂₄ is a substituent on the cyclohexyl group and represents ahydrogen atom, a CN group, a NO₂ group, a halogen atom, a CH₃ group, aC₂H₅ group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F₇ group;and r is an integer selected within a range indicated in the chemicalformula.)

More specifically, each precursor PHA can be more advantageouslyprepared by culturing a microorganism in a culture medium containingrespective raw material compounds, namely, for the precursor vinyl PHA,a combination of at least one ω-alkenoic acid represented by thechemical formula (24) and at least one compound represented by thechemical formula (25) or at least one ω-cyclohexylalkanoic acidrepresented by the chemical formula (26); and for the precursoralkoxycarbonyl PHA, a combination of at least one carboxylic acidmonoester compound represented by the chemical formula (49) and at leastone compound represented by the chemical formula (25) or at least oneω-cyclohexylalkanoic acid represented by the chemical formula (26), andfurther containing at least one of peptide, yeast extract, organic acidor salt thereof, amino acid or a salt thereof, sugar, and linearalkanoic acid with 4 to 12 carbon atoms or salt thereof.

As preferable nutrients to be added to the culture medium, the peptidebeing polypeptone; one or more organic acids selected from the group ofpyruvic acid, oxaloacetic acid, citric acid, isocitric acid,ketoglutaric acid, succinic acid, fumaric acid, malic acid, lactic acidand salts thereof; one or more amino acids selected from the group ofglutamic acid, aspartic acid and salts thereof; and one or more sugarsselected from the group of glyceraldehyde, erythrose, arabinose, xylose,glucose, galactose, mannose, fructose, glycerol, erythritol, xylitol,gluconic acid, glucuronic acid, galacturonic acid, maltose, sucrose andlactose.

In the producing method of the precursor PHA copolymer of the presentinvention, detailed microbial culture conditions are as follows.

The following necessary substrates and nutrients are added to aninorganic salt culture medium based on a phosphate buffer and anammonium salt or a nitrate salt.

The raw material compound for each precursor PHA, namely, for theprecursor vinyl PHA, a combination of at least an ω-alkenoic acidrepresented by the chemical formula (24) and at least a compoundrepresented by the chemical formula (25) or at least anω-cyclohexylalkanoic acid represented by the chemical formula (26); orfor the precursor alkoxycarbonyl PHA, a combination of at least acarboxylic acid monoester compound represented by the chemical formula(49) and at least a compound represented by the chemical formula (25) orat least an ω-cyclohexylalkanoic acid represented by the chemicalformula (26), is preferably contained in the culture medium in aproportion of 0.01 to 1% (w/v), further preferably 0.02 to 0.2%.

The aforementioned nutrients as a carbon source and a nitrogen sourcefor proliferation, and as an energy source for polyhydroxy alkanoateproduction are preferably added to the culture medium in a proportion of0.1 to 5% (v/v) per medium, more preferably 0.2 to 2%.

It can be employed any inorganic salt culture medium containing aphosphate salt and a nitrogen source such as an ammonium salt or anitrate salt, but the PHA productivity can be improved by regulating theconcentration of the nitrogen source.

The culture temperature can be any temperature at which themicroorganism can satisfactorily proliferate, and is usually within arange of 15 to 37° C., preferably 20 to 30° C.

The culture may be carried out by any culture method so long as themicroorganisms can proliferate and produce PHA, such as a liquid cultureor a solid culture. Also it may be batch culture, fed batch culture,semi-continuous culture or continuous culture. For example, for liquidbatch culture, the oxygen supply method may be shaking using a shakingflask or agitation aeration in a jar fermenter.

In order to make the microorganism produce and accumulate PHA, there canbe employed, in addition to the aforementioned method, a method oftransferring the cell, after sufficient proliferation, to a culturemedium limited in a nitrogen source such as ammonium chloride and tocontinue culture further in the presence of a compound being a substratefor the desired unit, thereby improving the productivity.

Thus the method for producing precursor vinyl PHA of the presentinvention may comprise the steps of: culturing a productionmicroorganism under the aforementioned conditions, and recoveringproduced PHA from the cells, the PHA copolymer produced by themicroorganism at least containing a 3-hydroxy-ω-alkenoic acid unitrepresented by the chemical formula (1), and a unit represented by thechemical formula (2) or an ω-cyclohexylalkanoic acid unit represented bythe chemical formula (3) in the molecule.

Also the method for producing precursor alkoxycarbonyl PHA of thepresent invention may comprise the steps of: culturing a productionmicroorganism under the aforementioned conditions, and recovering fromthe cells a polyhydroxy alkanoate copolymer produced by themicroorganism which at least contains a3-hydroxy-ω-alkoxycarbonylalkanoic acid unit represented by the chemicalformula (48), and a unit represented by the chemical formula (2) or anω-cyclohexylalkanoic acid unit represented by the chemical formula (3)in the molecule.

The object PHA can be recovered from the cells by an ordinarily employedmethod. For example, an extraction with an organic solvent such aschloroform, dichloromethane, ethyl acetate or acetone is most simple,but there may also be employed dioxane, tetrahydrofuran or acetonitrile.Also in a situation where an organic solvent is difficult to use, it isalso possible to physically break the cells, for example by treating thecells with a surfactant such as SDS, chemicals such as hypochlorous acidand EDTA, or with an enzyme such as lysozyme, or by ultrasonicdisruption, homogenizer disruption, pressure disruption, beads impulse,grinding or pounding or freeze-and-thawing, to remove cell componentsother than PHA and recover PHA.

A production microorganism to be employed in the production method ofthe present invention can be any microorganisms having an abilitymeeting the aforementioned conditions, but there are preferred thosebelonging to the Pseudomonas genus, and more preferably Pseudomonascichorii, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonasoleovorans, Pseudomonas aeruginosa, Pseudomonas stutzeri or Pseudomonasjessenii. More specific examples include Pseudomonas cichorii YN2 (FERMBP-7375), Pseudomonas cichorii H45 (FERM BP-7374), Pseudomonas jesseniiP161 (FERM BP-7376), and Pseudomonas putida P91 (FERM BP-7373). Thesefour types of strains are deposited on Nov. 20, 2000 at InternationalPatent Organism Depositary, National Institute of Bioscience andHuman-Technology, Agency of Industry Science and Technology (independentadministrative corporation), Tsukuba Central 6, 1-1, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken 305-8566, Japan, and described in the JapanesePatent Application Laid-Open No. 2002-80571.

In the present invention the methods for culture of the microorganism,PHA production and accumulation by the microorganism, and for PHArecovery from the cells are not limited to the methods explained above.

The following is a composition of an inorganic salt M9 culture mediumemployed in the method of the present invention.

[M9 culture medium] Na₂HPO₄ 6.3 KH₂PO₄ 3.0 NaCl 0.5 NH₄Cl 1.0 (in g/L;pH 7.0)

For satisfactory proliferation and resulting PHA production, theabove-mentioned inorganic culture medium has to be replenished with theessential trace elements by adding the following trace componentsolution by about 0.3% (v/v).

[Minor component solution] nytrilotriacetic acid 1.5; MgSO₄ 3.0; MnSO₄0.5; NaCl 1.0; FeSO₄ 0.1; CaCl₂ 0.1; CoCl₂ 0.1; ZnSO₄ 0.1; CuSO₄ 0.1;AlK(SO₄)₂ 0.1; H₃BO₃ 0.1; Na₂MoO₄ 0.1; NiCl₂ 0.1; (in g/L).

The polyhydroxy alkanoates synthesized by the aforementioned producingmethod, a polyhydroxy alkanoate copolymer including a unit representedby the chemical formula (1) and a unit represented by the chemicalformula (2) or a unit represented by the chemical formula (3) can beoxidized at the carbon-carbon double bond portion to give a polyhydroxyalkanoate copolymer including a unit represented by the chemical formula(19), and a unit represented by the chemical formula (2) or a unitrepresented by the chemical formula (3). For obtaining a carboxylic acidby oxidizing a carbon-carbon double bond with an oxidant, there areknown, for example, a method of utilizing a permanganate salt (J. Chem.Soc. Perkin. Trans. 1, 806(1973)); a method of utilizing a bichromatesalt (Org. Synth., 4, 698(1963)); a method of utilizing a periodate salt(J. Org. Chem., 46, 19(1981)); a method of utilizing nitric acid(Japanese Patent Application Laid-Open No. S59-190945); a method ofutilizing ozone (J. Am. Chem. Soc., 81, 4273(1959)) etc., and, onpolyhydroxy alkanoate, Macromolecular chemistry, 4, 289-293(2001)reports a method of obtaining a carboxylic acid by oxidizing thecarbon-carbon double bond at the end of the side chain of polyhydroxyalkanoate with potassium permanganate as an oxidant and under an acidiccondition. A similar method can be utilized also in the presentinvention.

The oxidant to be employed in the present invention, though notparticularly limited, is preferably a permanganate salt. Suchpermanganate salt to be employed as the oxidant is usually potassiumpermanganate. Since the oxidation reaction is a stoichiometric reaction,the amount of the permanganate salt is usually 1 molar equivalent ormore with respect to 1 mole of the unit represented by the chemicalformula (1), preferably 2 to 10 molar equivalents.

For executing the reaction under an acidic condition, there is usuallyemployed an inorganic acid such as sulfuric acid, hydrochloric acid,acetic acid or nitric acid, or an organic acid. However the use ofsulfuric acid, nitric acid or hydrochloric acid may cause cleavage of anester bond in the main chain of polyhydroxy alkanoate, thereby resultingin a decrease in the molecular weight. It is therefore preferable toemploy acetic acid. An amount of acid is usually within a range of 0.2to 2000 molar equivalents per 1 mole of the unit represented by thechemical formula (1), preferably 0.4 to 1000 molar equivalents. Anamount less than 0.2 molar equivalents results in a low yield, while anamount exceeding 2000 molar equivalents generates by-products bydecomposition with acid. Also a crown ether may be employed for thepurpose of accelerating the reaction. In this case, crown ether andpermanganate salt form a complex, thereby providing an effect ofincreasing the reaction activity. As the crown ether, there is generallyemployed dibenzo-18-crown-6-ether, dicyclo-18-crown-6-ether, or18-crown-6-ether. An amount of crown ether is generally within a rangeof 0.005 to 2.0 molar equivalents per 1 mole of permanganate salt,preferably 0.05 to 1.5 molar equivalents.

As a solvent to be employed in the oxidation reaction of the presentinvention, there may be employed any solvent inert to the reactionwithout particular limitation, for example water, acetone; an ether suchas tetrahydrofuran or dioxane; an aromatic hydrocarbon such as benzene;an aliphatic hydrocarbon such as hexane or heptane; or a halogenatedhydrocarbon such as methyl chloride, dichloromethane or chloroform.Among these solvents, in consideration of dissolving property for thepolyhydroxy alkanoate, a halogenated hydrocarbon such as methylchloride, dichloromethane or chloroform, or acetone is preferred.

In the aforementioned oxidation reaction of the present invention, aprecursor vinyl PHA, a permanganate salt and an acid may be introducedinto a solvent at a time from the beginning and reacted together, orthey may be added to the reaction system one by one continuously orintermittently to be reacted. Or first a permanganate alone is dissolvedor suspended in a solvent, followed by continuous or intermittentaddition of a polyhydroxyalkanoate and an acid to the reaction system,or first a polyhydroxyalkanoate alone is dissolved or suspended in asolvent, followed by continuous or intermittent addition of apermanganate and an acid to the reaction system. Further, first apolyhydroxyalkanoate and an acid are introduced into a solvent and thena permanganate is added to the reaction system continuously orintermittently to be reacted, or first permanganate and an acid areintroduced into a solvent and then polyhydroxyalkanoate is added to thereaction system continuously or intermittently, or first apolyhydroxyalkanoate and a permanganate are introduced into a solventand then an acid is added to the reaction system continuously andintermittently to be reacted.

A reaction temperature is selected generally within a range from −40 to40° C., preferably −10 to 30° C. A reaction time depends on astoichiometric ratio of the unit represented by the chemical formula (1)and permanganate salt and the reaction temperature, but is generallyselected within a range of 2 to 48 hours.

Also in the oxidation reaction of the present invention, in case R₂ inthe unit represented by the chemical formula (2) is a residuerepresented by the chemical formula (11), a sulfide bond therein may beconverted into a sulfoxide or a sulfone.

Next, there will be explained the producing method of the precursorester PHA of the present invention employing, as a starting material, apolyhydroxy alkanoate copolymer including a unit represented by thechemical formula (48), and a unit represented by a chemical formula (2)or a unit represented by a chemical formula (3).

A precursor ester PHA synthesized can provide the carboxyl PHA byhydrolysis in the presence of an acid or an alkali or hydrogenolysisincluding catalytic reduction of an ester bond portion shown in thechemical formula (48). Such method of hydrolysis in the presence of anacid or an alkali can be carried out by employing, in a water-miscibleorganic solvent such as methanol, ethanol, tetrahydrofuran, dioxane,dimethylformamide or dimethylsulfoxide, an aqueous solution or aninorganic acid such as hydrochloric acid, sulfuric acid, nitric acid orphosphoric acid; an organic acid such as trifluoroacetic acid,trichlorbacetic acid, p-toluenesulfonic acid or methanesulfonic acid; anaqueous caustic alkali such as sodium hydroxide or potassium hydroxide;an.aqueous solution or an alkali carbonate such as sodium carbonate orpotassium carbonate; or an alcoholic solution of a metal alkoxide suchas sodium methoxide or sodium ethoxide. The reaction temperature isselected ordinarily from 0 to 40° C., preferably from 0 to 30° C. Thereaction period is ordinarily selected from 0.5 to 48 hours. However, ahydrolysis with an acid or an alkali may also cause a cleavage of anester bonding of the main molecular chain, thereby resulting in adecrease in the molecular weight.

Also the method of obtaining a carboxylic acid by hydrogenolysisincluding catalytic reduction is carried out in the following manner.Catalytic reduction is carried out in a suitable solvent and within atemperature range from −20° C. to the boiling point of the used solvent,preferably from 0 to 50° C., by reacting hydrogen under a normalpressure or an elevated pressure in the presence of a reducing catalyst.Examples of the usable solvent include water, methanol, ethanol,propanol, ethyl acetate, diethyl ether, tetrahydrofuran, dioxane,benzene, toluene, dimethylformamide and pyridine. In consideration ofthe solubility, tetrahydrofuran, toluene or dimethylformamide isparticularly preferable. As the reducing catalyst, there can be employedpalladium, platinum or rhodium either singly or held on a carrier, orRaney nickel. However, the catalytic reduction may also cause cleavageof an ester bonding of the main molecular chain to decrease themolecular weight.

In the following, the present invention will be explained in moredetails by examples thereof. These examples represent examples of theoptimum embodiments of the present invention, but the present inventionis by no means limited by these examples.

EXAMPLES Example 1

0.5% of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-phenoxyvaleric acid, and 1 mmol/L of 10-undecenoic acid were dissolvedin 200 ml of an aforementioned M9 culture medium, which was placed in a200 ml shaking flask, then sterilized in an autoclave and cooled to theroom temperature. Then 2 ml of a culture liquid of Pseudomonas cichoriiYN2, shake cultured in advance in an M9 culture medium containing 0.5%of polypeptone for 8 hours at 30° C., was added to the prepared culturemedium, and culture was conducted for 64 hours at 30° C. After theculture, the cells were collected by centrifugation, washed withmethanol and dried. The dried cells, after weighing, were put inchloroform and stirred for 72 hours at 35° C. to extract a polymer. Thechloroform extract was filtered, then concentrated on an evaporator, anda solid precipitate formed by an addition of cold methanol was collectedand dried under a reduced pressure to obtain a desired polymer.

Structure of the obtained polymer was determined by ¹H-NMR (FT-NMR:Bruker DPX400; resonance frequency: 400 MHz; measured nucleus species:¹H; solvent: CDCl₃; reference: capillary-sealed TMS/CDCl₃; measurementtemperature: room temperature) and ¹³C-NMR (FT-NMR: Bruker DPX400;resonance frequency: 100 MHz; measured nucleus species: ¹³C; solvent:CDCl₃; reference: capillary-sealed TMS/CDCl₃; measurement temperature:room temperature).

FIG. 1 shows a ¹H-NMR spectrum of the obtained polymer. As a result, theobtained polymer was confirmed being a polyhydroxy alkanoate copolymerincluding a unit represented by the following chemical formula (50)(A:B+C+D: others (linear 3-hydroxyalkanoic acid with 4 to 12 carbonatoms and 3-hydroxylalk-5-enoic acid with 10 or 12 carbonatoms)=87:9:4). Also ¹³C-NMR confirmed presence of the unit B which is a3-hydroxy-10-undecenoic acid unit and both of the unit C which is a3-hydroxy-8-nonenoic acid unit and the unit D which is a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by gelpermeation chromatography (GPC) (Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, molecular weight converted intopolystyrene).

The obtained polymer dry weight (PDW) was 0.19 g/L and thenumber-averaged molecular weight was 30,000.

Example 2

A desired polymer was obtained in the same manner as in Example 1,except that 5-phenoxyvaleric acid employed in Example 1 was changed to4-phenoxybutyric acid.

Structure of the obtained polymer was determined by ¹H-NMR and ¹³C-NMR(FT-NMR: Bruker DPX400 as in Example 1. As a result, the obtainedpolymer was confirmed being a polyhydroxy alkanoate copolymer includingunits represented by the following chemical formula (51) (A:B+C+D:others (linear 3-hydroxyalkanoic acid with 4 to 12 carbon atoms and3-hydroxylalk-5-enoic acid with 10 or 12 carbon atoms)=72:11:15). Also¹³C-NMR confirmed the presence of the unit B which is a3-hydroxy-10-undecenoic acid unit and both of the unit C which is a3-hydroxy-8-nonenoic acid unit and the unit D which is a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The obtained polymer weighed (PDW) 0.05 g/L and a number-averagedmolecular weight was 25,000.

Example 3

A desired polymer was obtained in the same manner as in Example 1,except that 5-phenoxyvaleric acid employed in Example 1 was changed to4-cyclohexylbutyric acid.

Structure of the obtained polymer obtained by ¹H-NMR and ¹³C-NMR as inExample 1 was determined to confirm that the polyhydroxy alkanoatecopolymer includes units represented by the following chemical formula(52) (A+others (linear 3-hydroxyalkanoic acid with 4 to 12 carbon atomsand 3-hydroxylalk-5-enoic acid with 10 or 12 carbon atoms):B+C+D=89:11). Also ¹³C-NMR confirmed the presence of the unit B being a3-hydroxy-10-undecenoic acid unit and both of the unit C being a3-hydroxy-8-nonenoic acid unit and the unit D being a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The obtained polymer weighed (PDW) 0.52 g/L and the number-averagedmolecular weight was 154,000.

Example 4

A desired polymer was obtained in the same manner as in Example 3,except that polypeptone employed in Example 3 was changed to yeastextract.

Structure of the obtained polymer was determined by ¹H-NMR and ¹³C-NMRas in Example 1 to confirm the polymer being a polyhydroxy alkanoatecopolymer including units represented by the following chemical formula(52) (A+others (linear 3-hydroxyalkanoic acid with 4 to 12 carbon atomsand 3-hydroxylalk-5-enoic acid with 10 or 12 carbon atoms):B+C+D=85:15). Also ¹³C-NMR confirmed the presence of the unit B is a3-hydroxy-10-undecenoic acid unit and both of the unit C being a3-hydroxy-8-nonenoic acid unit and the unit D being a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The obtained polymer weighed (PDW) 0.45 g/L and the number-averagedmolecular weight was 132,000.

Example 5

A polymer was obtained in the same manner as in Example 3, except thatthe strain YN2 employed in Example 3 was replaced by Pseudomonascichorii H45 and polypeptone was changed to glucose. Structure of theobtained polymer was determined by ¹H-NMR and ¹³C-NMR as in Example 1 toconfirm the polymer being a polyhydroxy alkanoate copolymer includingunits represented by the following chemical formula (52) (A+others(linear 3-hydroxyalkanoic acid with 4 to 12 carbon atoms and3-hydroxylalk-5-enoic acid with 10 or 12 carbon atoms): B+C+D=83:17).Also ¹³C-NMR confirmed the presence of the unit B being a3-hydroxy-10-undecenoic acid unit and both of the unit C being a3-hydroxy-8-nonenoic acid unit and the unit D being a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The obtained polymer weighed (PDW) 0.41 g/L and the number-averagedmolecular weight was 164,000.

Example 6

A polymer was obtained in the same manner as in Example 3, except thatthe strain YN2 employed in Example 3 was replaced by Pseudomonascichorii H45 and polypeptone was changed to sodium pyruvate. A structuredetermination of the obtained polymer was conducted by ¹H-NMR and¹³C-NMR as in Example 1 to confirm the polymer being a polyhydroxyalkanoate copolymer including units represented by the followingchemical formula (52) (A+others (linear 3-hydroxyalkanoic acid with 4 to12 carbon atoms and 3-hydroxylalk-5-enoic acid with 10 or 12 carbonatoms): B+C+D=87:13). Also ¹³C-NMR confirmed the presence of the unit Bbeing a 3-hydroxy-10-undecenoic acid unit and the unit C being a3-hydroxy-8-nonenoic acid unit and the unit D being a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The weight of the obtained polymer (PDW) was 0.28 g/L and thenumber-averaged molecular weight was 156,000.

Example 7

A polymer was obtained in the same manner as in Example 3, except thatthe strain YN2 employed in Example 3 was replaced by Pseudomonasjessenii P161 and polypeptone was changed to sodium glutamate. Structuredetermination of the obtained polymer was conducted by ¹H-NMR and¹³C-NMR as in Example 1 to confirm the polymer being a polyhydroxyalkanoate copolymer including units represented by the followingchemical formula (52) (A+others (linear 3-hydroxyalkanoic acid with 4 to12 carbon atoms and 3-hydroxylalk-5-enoic acid with 10 or 12 carbonatoms): B+C+D=88:12). Also ¹³C-NMR confirmed the presence of the unit Bbeing a 3-hydroxy-10-undecenoic acid unit and both of the unit C being a3-hydroxy-8-nonenoic acid unit and the unit D being a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The weight of the obtained polymer (PDW) was 0.38 g/L and thenumber-averaged molecular weight of 145,000.

Example 8

A polymer was obtained in the same manner as in Example 3, except thatthe strain YN2 employed in Example 3 was replaced by Pseudomonasjessenii P161 and 0.5% polypeptone was changed to 0.1% of nonanic acid.The structure determination of the obtained polymer was conducted by¹H-NMR and ¹³C-NMR as in Example 1 to confirm the polymer being apolyhydroxy alkanoate copolymer including units represented by thefollowing chemical formula (52) (A+others (linear 3-hydroxyalkanoic acidwith 4 to 12 carbon atoms and 3-hydroxylalk-5-enoic acid with 10 or 12carbon atoms): B+C+D=80:20). Also ¹³C-NMR confirmed the presence of theunit B being a 3-hydroxy-10-undecenoic acid unit and both of the unit Cbeing a 3-hydroxy-8-nonenoic acid unit and the unit D being a3-hydroxy-6-heptenoic acid unit, but the ratio of the units B, C and Dwas not determined.

The molecular weight of the obtained polymer was measured by GPC as inExample 1.

The weight of the obtained polymer (PDW) was 0.18 g/L and thenumber-averaged molecular weight was 132,000.

Example 9

Twenty 200 ml shaking flasks were prepared, into which 0.5% ofpolypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-phenoxyvaleric acid, and 1 mmol/L of 10-undecenoic acid dissolved in200 ml of an aforementioned M9 culture medium was placed, thensterilized in an autoclave and cooled to the room temperature. Then 2 mlof a culture liquid of Pseudomonas cichorii YN2, shake cultured inadvance in an M9 culture medium containing 0.5% of polypeptone for 8hours at 30° C., was added to each flask, and culture was conducted for64 hours at 30° C. After the culture, all cells were collected bycentrifugation, washed with methanol and dried. The dried cells, afterweighing, were put in chloroform and stirred for 72 hours at 35° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated on an evaporator, and a solid precipitate formed by anaddition of cold methanol was collected and dried under a reducedpressure to obtain a desired polymer.

The obtained PHA polymer weighed 1528 mg (dry weight) in the presentexample.

The average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=104000 and aweight-averaged molecular weight Mw=231000. The structure of theobtained polymer was determined by ¹H-NMR and ¹³C-NMR as in Example 1.

As a result, confirmed was a polyhydroxy alkanoate copolymer including,as monomer units, 3-hydroxy-5-phenoxyvaleric acid represented by thefollowing chemical formula (53), 3-hydroxy-10-undecenoic acidrepresented by a chemical formula (5), 3-hydroxy-8-nonenoic acidrepresented by a chemical formula (6) and 3-hydroxy-6-heptenoic acidrepresented by a chemical formula (7).

The proportion of such units confirmed by ¹H-NMR was: 69 mol % of3-hydroxy-5-phenoxyvaleric acid, 23 mol % of three units of3-hydroxy-10-undecenoic acid, 3-hydroxy-8-nonenoic acid and3-hydroxy-6-heptenoic acid in total, and 8 mol % of others (linear3-hydroxyalkanoic acids of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacids with 10 or 12 carbon atoms).

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

303 mg of polyhydroxy alkanoate were charged in a 200-ml eggplant-shapedflask and were dissolved by adding 20 ml of dichlordmethane. Thesolution was placed in an iced bath, and 3 ml of acetic acid and 300 mgof 18-crown-6-ether were added and agitated. Then, in an iced bath, 241mg of potassium permanganate were slowly added and an agitation wascarried out for 20 hours at the room temperature. After the reaction, 50ml of water and 500 mg of sodium bisulfite were added. Then the liquidwas brought to pH=1 by 1.0 N hydrochloric acid. After dichloromethane inthe mixed solvent was distilled off in an evaporator, a polymer in thesolution was recovered. The polymer was recovered by washing with 100 mlof methanol and washing three times with 100 ml of purified water. Adrying under a reduced pressure provided 247 mg of the desired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=29400 and aweight-averaged molecular weight Mw=102800.

A structure determination of the obtained polymer carried out by ¹H-NMRand ¹³C-NMR as in Example 1 confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenoxyvaleric acid representedby the following chemical formula (53), 3-hydroxy-9-carboxynonanoic acidrepresented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoicacid represented by a chemical formula (55) and3-hydroxy-5-carboxyvaleric acid represented by a chemical formula (56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

50 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 3.5 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 2 ml of a 0.63 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Tokyo Kasei Kogyo Co.)and was agitated for 30 minutes at the room temperature. After thereaction, the solvent was distilled off in an evaporator to recover apolymer. The polymer was recovered by washing with 50 ml of methanol. Adrying under a reduced pressure provided 49 mg of PHA.

NMR analysis as in Example 1 confirmed a proportion of the units inwhich 3-hydroxy-5-phenoxyvaleric acid was present by 83 mol %, a sum ofthree units of 3-hydroxy-9-carboxynonanoic acid,3-hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by8 mol %, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbonatoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 9 mol%.

Example 10

There were prepared twenty 500-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of4-cyclohexylbutyric acid, and 3 mmol/L of 10-undecenoic acid weredissolved in 200 ml of an aforementioned M9 culture medium, which wasplaced in a 500 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 2 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 25° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1433 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=143000 and aweight-averaged molecular weight Mw=458000.

A structure of the obtained PHA was determined by a NMR analysis as inExample 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-cyclohexylbutyric acidrepresented by the following chemical formula (57),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by37 mol %, and 3-hydroxy-4-cyclohexylbutyric acid and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 63 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

301 mg of polyhydroxy alkanoate were charged in a 200-ml eggplant-shapedflask and were dissolved by adding 20 ml of dichloromethane. Thesolution was placed in an iced bath, and 3 ml of acetic acid and 541 mgof 18-crown-6-ether were added and agitated. Then, in an iced bath, 430mg of potassium permanganate were slowly added and an agitation wascarried out for 20 hours at the room temperature. After the reaction, 50ml of water and 1000 mg of sodium bisulfite were added. Then the liquidwas brought to pH=1 by 1.0 N hydrochloric acid. After dichloromethane inthe mixed solvent was distilled off in an evaporator, a polymer in thesolution was recovered. The polymer was recovered by washing with 100 mlof methanol and washing three times with 100 ml of purified water. Adrying under a reduced pressure provided 184 mg of the desired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=111800 and aweight-averaged molecular weight Mw=272800.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-4-cyclohexylvaleric acidrepresented by the following chemical formula (57),3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54),3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55)and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula(56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.4 ml ofmethanol. The solution was added with 0.9 ml of a 0.63 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Tokyo Kasei Kogyo Co.)and was agitated for 30 minutes at the room temperature. After thereaction, the solvent was distilled off in an evaporator to recover apolymer. The polymer was recovered by washing with 50 ml of methanol. Adrying under a reduced pressure provided 31 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, there wasconfirmed a proportion of the units in which a sum of three units of3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoic acid and3-hydroxy-5-carboxyvaleric acid was present by 9 mol %, and3-hydroxy-4-cyclohexyl butyric acid and others (linear 3-hydroxyalkanoicacid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoic acid with 10 or 12carbon atoms) by 91 mol %.

Example 11

There were prepared three 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 4.8 mmol/L of5-(phenylsulfanyl)valeric acid, and 2 mmol/L of 10-undecenoic acid weredissolved in 1000 ml of an aforementioned M9 culture medium, which wasplaced in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 38 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 25 hoursat 35° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1934 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=150000 and aweight-averaged molecular weight Mw=430000.

A structure of the obtained PHA was determined by a NMR analysis as inExample 1. An obtained ¹H-NMR spectrum is shown in FIG. 3.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(phenylsulfanyl)valeric acidrepresented by the following chemical formula (58),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, where3-hydroxy-5-(phenylsulfanyl)valeric acid was present by 78 mol %, a sumof three units of 3-hydroxy-10-undecenoic acid, 3-hydroxy-8-nonenoicacid and 3-hydroxy-6-heptenoic acid by 19 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 3 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction. 302 mg of polyhydroxy alkanoate were charged in a 200-mleggplant-shaped flask and were dissolved by adding 20 ml ofdichloromethane. The solution was placed in an iced bath, and 3 ml ofacetic acid and 1154 mg of 18-crown-6-ether were added and agitated.Then, in an iced bath, 917 mg of potassium permanganate were slowlyadded and an agitation was carried out for 19 hours at the roomtemperature. After the reaction, 50 ml of water and 3010 mg of sodiumbisulfite were added. Then the liquid was brought to pH=1 by 1.0 Nhydrochloric acid. After dichloromethane in the mixed solvent wasdistilled off in an evaporator, a polymer in the solution was recovered.The polymer was recovered by washing with 100 ml of methanol and washingthree times with 100 ml of purified water. A drying under a reducedpressure provided 311 mg of the desired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=62000 and aweight-averaged molecular weight Mw=260000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1. An obtained ¹H-NMRspectrum is shown in FIG. 4.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(phenylsulfonyl)valeric acidrepresented by the following chemical formula (59),3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54),3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55)and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula(56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.5 ml of a 2 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Aldrich Inc.) and wasagitated for 30 minutes at the room temperature. After the reaction, thesolvent was distilled off in an evaporator to recover a polymer. Thepolymer was recovered by washing with 50 ml of methanol. A drying undera reduced pressure provided 31 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-(phenylsulfonyl)valeric acid was present by 89 mol %, a sumof three units of 3-hydroxy-9-carboxynonanoic acid,3-hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by8 mol %, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbonatoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 3 mol%.

Example 12

There were prepared three 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-phenylvaleric acid, and 1.5 mmol/L of 10-undecenoic acid weredissolved in 1000 ml of an aforementioned M9 culture medium, which wasplaced in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 35° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1533 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=72000 and aweight-averaged molecular weight Mw=170000.

A structure of the obtained PHA was determined by a NMR analysis as inExample 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60), 3-hydroxy-10-undecenoic acidrepresented by a chemical formula (5), 3-hydroxy-8-nonenoic acidrepresented by a chemical formula (6) and 3-hydroxy-6-heptenoic acidrepresented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by12 mol %, 3-hydroxy-5-phenylvaleric acid by 85 mol % and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 3 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

1002 mg of polyhydroxy alkanoate were charged in a 500-mleggplant-shaped flask and were dissolved by adding 60 ml ofdichloromethane. The solution was placed in an iced bath, and 10 ml ofacetic acid and 537 mg of 18-crown-6-ether were added and agitated.Then, in an iced bath, 429 mg of potassium permanganate were slowlyadded and an agitation was carried out for 2 hours in an iced bath and18 hours at the room temperature. After the reaction, 40 ml of ethylacetate, 30 ml of water and 1000 mg of sodium bisulfite were added. Thenthe liquid was brought to pH=1 by 1.0 N hydrochloric acid. A polymer wasrecovered by extraction followed by distilling off of the solvent. Thepolymer was recovered by washing with 300 ml of purified water, thenwith 200 ml of methanol, three times with 200 ml of purified water andfinally with 200 ml of methanol. The obtained polymer was dissolved in10 ml of tetrahydrofuran and dialyzed for 1 day with a dialysis film(manufactured by Spectrum Inc., Stectra/Por Standard RegeneratedCellulose Dialysis Membrane 3), in a 1-L beaker containing 500 ml ofmethanol. The polymer present in the dialysis film was recovered anddried under a reduced pressure to obtain 953 mg of a desired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=43000 and aweight-averaged molecular weight Mw=94000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60), 3-hydroxy-9-carboxynonanoic acidrepresented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoicacid represented by a chemical formula (55) and3-hydroxy-5-carboxyvaleric acid represented by a chemical formula (56).

Also a proportion of the units of the obtained PHA was calculated by a.methylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.5 ml of a 2 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Aldrich Inc.) and wasagitated for 30 minutes at the room temperature. After the reaction, thesolvent was distilled off in an evaporator to recover a polymer. Thepolymer was recovered by washing with 50 ml of methanol. Drying under areduced pressure provided 30 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-phenylvaleric acid was present by 86 mol %, a sum of threeunits of 3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoicacid and 3-hydroxy-5-carboxyvaleric acid by 9 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 5 mol %.

Example 13

500 mg of polyhydroxy alkanoate copolymer, including3-hydroxy-5-phenylvaleric acid represented by the following chemicalformula (60), 3-hydroxy-10-undecenoic acid represented by a chemicalformula (5), 3-hydroxy-8-nonenoic acid represented by a chemical formula(6) and 3-hydroxy-6-heptenoic acid represented by a chemical formula (7)as monomer units used for the bacterial production in Example 12 werechanged in a 500-ml three-necked flask, and were suspended by adding 150ml of distilled water containing 50 ppm of hydrogen peroxide. Ozone wasblown in with a rate of 50 mg/hr and the mixture was agitated for 3hours at the room temperature.

After the reaction, the reaction liquid was filtered to recover apolymer. The polymer was resuspended in distilled water, and centrifugedto wash off remaining hydrogen peroxide. The obtained polymer wasfurther dissolved in 5 ml of tetrahydrofuran and dialyzed for 1 day witha dialysis film (manufactured by Spectrum Inc., Stectra/Por StandardRegenerated Cellulose Dialysis Membrane 3), in a 300-ml beakercontaining 250 ml of methanol. The polymer present in the dialysis filmwas recovered and dried under a reduced pressure to obtain 450 mg of adesired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Tso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=35000 and aweight-averaged molecular weight Mw=72000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60), 3-hydroxy-9-carboxynonanoic acidrepresented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoicacid represented by a chemical formula (55) and3-hydroxy-5-carboxyvaleric acid represented by a chemical formula (56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.3 ml of a 2 mol/L hexanesolution of trimethylsilyldiazomethane in hexane (supplied by AldrichInc.) and was agitated for 30 minutes at the room temperature. After thereaction, the solvent was distilled off in an evaporator to recover apolymer. The polymer was recovered by washing with 50 ml of methanol. Adrying under a reduced pressure provided 30 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, there wasconfirmed a proportion of the units in which 3-hydroxy-5-phenylvalericacid was present by 85 mol %, a sum of three units of3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoic acid and3-hydroxy-5-carboxyvaleric acid by 10 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 5 mol %.

Example 14

There were prepared five 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-(4-vinylphenyl)valeric acid, and 1 mmol/L of 10-undecenoic acid weredissolved in 1000 ml of an aforementioned M9 culture medium, which wasplaced in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 25° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1097 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=70000 and aweight-averaged molecular weight Mw=150000.

A structure of the obtained PHA was determined by a NMR analysis as inExample 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(4-vinylphenyl)valeric acidrepresented by the following chemical formula (61),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by9 mol %, 3-hydroxy-5-(4-vinylphenyl)valeric acid by 84 mol % and others(linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 7 mol %.

Example 15

There were prepared twenty 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-benzoylvaleric acid, and 1 mmol/L of 10-undecenoic acid were dissolvedin 1000 ml of an aforementioned M9 culture medium, which was placed in a2000 ml shake flask, then sterilized in an autoclave and cooled to theroom temperature. Then 10 ml of a culture liquid of Pseudomonas cichoriiYN2 strain, shake cultured in advance in an M9 culture medium containing0.5% of polypeptone for 8 hours, was added to each prepared culturemedium, and culture was conducted for 60 hours at 30° C. After theculture, the culture liquids were united, and the cells were recoveredby centrifuging, rinsed with methanol and dried. The dried cells, afterweighing, were agitated with chloroform for 72 hours at 25° C. toextract a polymer. The chloroform extract was filtered with a 0.45 μmmembrane filter, then concentrated in an evaporator, and the polymer wasrecovered by a reprecipitation in cold methanol. A desired polymer wasthen obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1027 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=120000 and aweight-averaged molecular weight Mw=370000.

A structure of the obtained PHA was determined by a NMR analysis as inExample 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-benzoylvaleric acid representedby the following chemical formula (62), 3-hydroxy-10-undecenoic acidrepresented by a chemical formula (5), 3-hydroxy-8-nonenoic acidrepresented by a chemical formula (6) and 3-hydroxy-6-heptenoic acidrepresented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by11 mol %, 3-hydroxy-5-benzoylvaleric acid by 82 mol % and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 7 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

1003 mg of polyhydroxy alkanoate were charged in a 500-mleggplant-shaped flask and were dissolved by adding 60 ml ofdichloromethane. The solution was placed in an iced bath, and 10 ml ofacetic acid and 410 mg of 18-crown-6-ether were added and agitated.Then, in an iced bath, 327 mg of potassium permanganate wereslowly-added and an agitation was carried out for 2 hours in an icedbath and 18 hours at the room temperature. After the reaction, 100 ml ofwater and 1000 mg of sodium bisulfite were added. Then the liquid wasbrought to pH=1 by 1.0 N hydrochloric acid. After dichloromethane in themixed solvent was distilled off in an evaporator, a polymer in thesolution was recovered. The polymer was recovered by washing with 200 mlof purified water, then with 200 ml of methanol, three times with 200 mlof purified water and finally with 200 ml of methanol. The obtainedpolymer was dissolved in 10 ml of tetrahydrofuran and dialyzed for 1 daywith a dialysis film (manufactured by Spectrum Inc., Stectra/PorStandard Regenerated Cellulose Dialysis Membrane 3), in a 1-L beakercontaining 500 ml of methanol. The polymer present in the dialysis filmwas recovered and dried under a reduced pressure to obtain 948 mg of adesired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=76000 and aweight-averaged molecular weight Mw=235000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-benzoylvaleric acid representedby the following chemical formula (62), 3-hydroxy-9-carboxynonanoic acidrepresented by a chemical formula (54), 3-hydroxy-7-carboxyheptanoicacid represented by a chemical formula (55) and3-hydroxy-5-carboxyvaleric acid represented by a chemical formula (56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.3 ml of a 2.0 mol/L hexanesolution of trimethylsilyldiazomethane in hexane (supplied by AldrichInc.) and was agitated for 30 minutes at the room temperature. After thereaction, the solvent was distilled off in an evaporator to recover apolymer. The polymer was recovered by washing with 50 ml of methanol. Adrying under a reduced pressure provided 29 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-benzoylvaleric acid was present by 84 mol %, a sum of threeunits of 3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoicacid and 3-hydroxy-5-carboxyvaleric acid by 9 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 7 mol %.

Example 16

There were prepared ten 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-[(phenylmethyl)sulfanyl]valeric acid, and 1.5 mmol/L of 10-undecenoicacid were dissolved in 1000 ml of an aforementioned M9 culture medium,which was placed in a 2000 ml shake flask, then sterilized in anautoclave and cooled to the room temperature. Then 10 ml of a cultureliquid of Pseudomonas cichorii YN2 strain, shake cultured in advance inan M9 culture medium containing 0.5% of polypeptone for 8 hours, wasadded to each prepared culture medium, and culture was conducted for 60hours at 30° C. After the culture, the culture liquids were united, andthe cells were recovered by centrifuging, rinsed with methanol anddried. The dried cells, after weighing, were agitated with chloroformfor 72 hours at 25° C. to extract a polymer. The chloroform extract wasfiltered with a 0.45 μm membrane filter, then concentrated in anevaporator, and the polymer was recovered by a reprecipitation in coldmethanol. A desired polymer was then obtained by drying under a reducedpressure.

According to a weighing of the obtained polymer, 1714 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=110000 and aweight-averaged molecular weight Mw=380000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under conditions similar to those in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-[(phenylmethyl)sulfanyl]valericacid represented by the following chemical formula (63),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by12 mol %, 3-hydroxy-5-[(phenylmethyl)sulfanyl]valeric acid by 80 mol %and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 8 mol %.

Example 17

There were prepared three 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-(2-thienyl)valeric acid, and 1.5 mmol/L of 10-undecenoic acid weredissolved in 1000 ml of an aforementioned M9 culture medium, which wasplaced in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 25° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1171 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=74000 and aweight-averaged molecular weight Mw=180000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under conditions similar to those in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(2-thienyl)valeric acidrepresented by the following chemical formula (64),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by12 mol %, 3-hydroxy-5-(2-thienyl)valeric acid by 85 mol % and others(linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 3 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

1001 mg of polyhydroxy alkanoate were charged in a 500-mleggplant-shaped flask and were dissolved by adding 60 ml ofdichloromethane. The solution was placed in an iced bath, and 10 ml ofacetic acid and 527 mg of 18-crown-6-ether were added and agitated.Then, in an iced bath, 420 mg of potassium permanganate were slowlyadded and an agitation was carried out for 2 hours in an iced bath and18 hours at the room temperature. After the reaction, 100 ml of waterand 1000 mg of sodium bisulfite were added. Then the liquid was broughtto pH=1 by 1.0 N hydrochloric acid. After dichloromethane in the mixedsolvent was distilled off in an evaporator, a polymer in the solutionwas recovered. The polymer was recovered by washing with 200 ml ofpurified water, then with 200 ml of methanol, three times with 200 ml ofpurified water and finally with 200 ml of methanol. The obtained polymerwas dissolved in 10 ml of tetrahydrofuran and dialyzed for 1 day with adialysis film (manufactured by Spectrum Inc., Stectra/Por StandardRegenerated Cellulose Dialysis Membrane 3), in a 1-L beaker containing500 ml of methanol. The polymer present in the dialysis film wasrecovered and dried under a reduced pressure to obtain 946 mg of adesired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=45000 and aweight-averaged molecular weight Mw=95000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(2-thienyl)valeric acidrepresented by the following chemical formula (64),3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54),3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55)and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula(56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.3 ml of a 2.0 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Aldrich Inc.) and wasagitated for 30 minutes at the room temperature. After the reaction, thesolvent was distilled off in an evaporator to recover a polymer. Thepolymer was recovered by washing with 50 ml of methanol. A drying undera reduced pressure provided 30 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-(2-thienyl)valeric acid was present by 85 mol %, a sum ofthree units of 3-hydroxy-9-carboxynonanoic acid,3-hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by10 mol %, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbonatoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 5 mol%.

Example 18

There were prepared three 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-(2-thienylsulfanyl)valeric acid, and 1 mmol/L of 10-undecenoic acidwere dissolved in 1000 ml of an aforementioned M9 culture medium, whichwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 25° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1257 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=68000 and aweight-averaged molecular weight Mw=160000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(2-thienylsulfanyl)valeric acidrepresented by the following chemical formula (65),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by9 mol %, 3-hydroxy-5-(2-thienylsulfanyl)valeric acid by 84 mol % andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 7 mol %.

Example 19

There were prepared ten 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-(2-thienylcarbonyl)valeric acid, and 1 mmol/L of 10-undecenoic acidwere dissolved in 1000 ml of an aforementioned M9 culture medium, whichwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 25° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1251 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=75000 and aweight-averaged molecular weight Mw=180000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under conditions similar to those in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(2-thienylcarbonyl)valeric acidrepresented by the following chemical formula (66),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by10 mol %, 3-hydroxy-5-(2-thienylcarbonyl)valeric acid by 81 mol % andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 9 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

999 mg of polyhydroxy alkanoate were charged in a 500-ml eggplant-shapedflask and were dissolved by adding 60 ml of dichloromethane. Thesolution was placed in an iced bath, and 10 ml of acetic acid and 382 mgof 18-crown-6-ether were added and agitated. Then, in an iced bath, 304mg of potassium permanganate were slowly added and an agitation wascarried out for 2 hours in an iced bath and 18 hours at the roomtemperature. After the reaction, 100 ml of water and 1000 mg of sodiumbisulfite were added. Then the liquid was brought to pH=1 by 1.0 Nhydrochloric acid. After dichloromethane in the mixed solvent wasdistilled off in an evaporator, a polymer in the solution was recovered.The polymer was recovered by washing with 200 ml of purified water, thenwith 200 ml of methanol, three times with 200 ml of purified water andfinally with 200 ml of methanol. The obtained polymer was dissolved in10 ml of tetrahydrofuran and dialyzed for 1 day with a dialysis film(manufactured by Spectrum Inc., Stectra/Por Standard RegeneratedCellulose Dialysis Membrane 3), in a 1-L beaker containing 500 ml ofmethanol. The polymer present in the dialysis film was recovered anddried under a reduced pressure to obtain 935 mg of a desired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=45000 and aweight-averaged molecular weight Mw=99000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(2-thienylcarbonyl)valeric acidrepresented by the following chemical formula (66),3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54),3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55)and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula(56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

31 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.3 ml of a 2.0 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Aldrich Inc.) and wasagitated for 30 minutes at the room temperature. After the reaction, thesolvent was distilled off in an evaporator to recover a polymer. Thepolymer was recovered by washing with 50 ml of methanol. A drying undera reduced pressure provided 30 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-(2-thienylcarbonyl)valeric acid was present by 83 mol %, asum of three units of 3-hydroxy-9-carboxynonanoic acid,3-hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by7 mol %, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbonatoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 10mol %.

Example 20

There were prepared fifteen 2000-ml shake flasks, and, in each, 0.5 wt.% of polypeptone (supplied by Wako Pure Chemical Co.), 6 mmol/L of5-[(phenylmethyl)oxy]valeric acid, and 1 mmol/L of 10-undecenoic acidwere dissolved in 1000 ml of an aforementioned M9 culture medium, whichwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 10 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours, was added toeach prepared culture medium, and culture was conducted for 60 hours at30° C. After the culture, the culture liquids were united, and the cellswere recovered by centrifuging, rinsed with methanol and dried. Thedried cells, after weighing, were agitated with chloroform for 72 hoursat 25° C. to extract a polymer. The chloroform extract was filtered witha 0.45 μm membrane filter, then concentrated in an evaporator, and thepolymer was recovered by a reprecipitation in cold methanol. A desiredpolymer was then obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1348 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=79000 and aweight-averaged molecular weight Mw=190000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under conditions similar to those in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-[(phenylmethyl)oxy]valeric acidrepresented by the following chemical formula (67),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of 'such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by10 mol %, 3-hydroxy-5-[(phenylmethyl)oxy]valeric acid by 82 mol % andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 8 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

1004 mg of polyhydroxy alkanoate were charged in a 500-mleggplant-shaped flask and were dissolved by adding 60 ml ofdichloromethane. The solution was placed in an iced bath, and 10 ml ofacetic acid and 389 mg of 18-crown-6-ether were added and agitated.Then, in an iced bath, 310 mg of potassium permanganate were slowlyadded and an agitation was carried out for 2 hours in an iced bath and18 hours at the room temperature. After the reaction, 100 ml of waterand 1000 mg of sodium bisulfite were added. Then the liquid was broughtto pH=1 by 1.0 N hydrochloric acid. After dichloromethane in the mixedsolvent was distilled off in an evaporator, a polymer in the solutionwas recovered. The polymer was recovered by washing with 200 ml ofpurified water, then with 200 ml of methanol, three times with 200 ml ofpurified water and finally with 200 ml of methanol. The obtained polymerwas dissolved in 10 ml of tetrahydrofuran and dialyzed for 1 day with adialysis film (manufactured by Spectrum Inc., Stectra/Por StandardRegenerated Cellulose Dialysis Membrane 3), in a 1-L beaker containing500 ml of methanol. The polymer present in the dialysis film wasrecovered and dried under a reduced pressure to obtain 940 mg of adesired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=48000 and aweight-averaged molecular weight Mw=106000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-[(phenylmethyl)oxy]valeric acidrepresented by the following chemical formula (67),3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54),3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55)and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula(56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.3 ml of a 2.0 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Aldrich Inc.) and wasagitated for 30 minutes at the room temperature. After the reaction, thesolvent was distilled off in an evaporator to recover a polymer. Thepolymer was recovered by washing with 50 ml of methanol. A drying undera reduced pressure provided 29 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-[(phenylmethyl)oxy]valeric acid was present by 84 mol %, asum of three units of 3-hydroxy-9-carboxynonanoic acid,3-hydroxy-7-carboxyheptanoic acid and 3-hydroxy-5-carboxyvaleric acid by8 mol %, and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbonatoms and 3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 8 mol%.

Example 21

There were prepared five 2000-ml shake flasks, and, in each, 0.5 wt. %of polypeptone (supplied by Wako Pure Chemical Co.), 3 mmol/L of5-phenoxyvaleric acid, 3 mmol/L of 5-cyclohexylvaleric acid and 1 mmol/Lof 10-undecenoic. acid were dissolved in 1000 ml of an aforementioned M9culture medium, which was placed in a 2000 ml shake flask, thensterilized in an autoclave and cooled to the room temperature. Then 10ml of a culture liquid of Pseudomonas cichorii YN2 strain, shakecultured in advance in an M9 culture medium containing 0.5% ofpolypeptone for 8 hours, was added to each prepared culture medium, andculture was conducted for 60 hours at 30° C. After the culture, theculture liquids were united, and the cells were recovered bycentrifuging, rinsed with methanol and dried. The dried cells, afterweighing, were agitated with chloroform for 72 hours at 25° C. toextract a polymer. The chloroform extract was filtered with a 0.45 μmmembrane filter, then concentrated in an evaporator, and the polymer wasrecovered by a reprecipitation in cold methanol. A desired polymer wasthen obtained by drying under a reduced pressure.

According to a weighing of the obtained polymer, 1285 mg (dry weight) ofPHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=86000 and aweight-averaged molecular weight Mw=230000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under conditions similar to those in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenoxyvaleric acid representedby the following chemical formula (53), 3-hydroxy-5-cyclohexylvalericacid represented by the following chemical formula (68),3-hydroxy-10-undecenoic acid represented by a chemical formula (5),3-hydroxy-8-nonenoic acid represented by a chemical formula (6) and3-hydroxy-6-heptenoic acid represented by a chemical formula (7).

Also a proportion of such units was confirmed by ¹H-NMR spectrum, wherea sum of three units of 3-hydroxy-10-undecenoic acid,3-hydroxy-8-nonenoic acid and 3-hydroxy-6-heptenoic acid was present by7 mol %, 3-hydroxy-5-phenoxyvaleric acid by 48 mol %,3-hydroxy-5-cyclohexylvaleric acid by 41 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 4 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

1002 mg of polyhydroxy alkanoate were charged in a 500-mleggplant-shaped flask and were dissolved by adding 60 ml ofdichloromethane. The solution was placed in an iced bath, and 10 ml ofacetic acid and 288 mg of 18-crown-6-ether were added and agitated.Then, in an iced bath, 230 mg of potassium permanganate were slowlyadded and an agitation was carried out for 2 hours in an iced bath and18 hours at the room temperature. After the reaction, 100 ml of waterand 1000 mg of sodium bisulfite were added. Then the liquid was broughtto pH=1 by 1.0 N hydrochloric acid. After dichloromethane in the mixedsolvent was distilled off in an evaporator, a polymer in the solutionwas recovered. The polymer was recovered by washing with 200 ml ofpurified water, then with 200 ml of methanol, three times with 200 ml ofpurified water and finally with 200 ml of methanol. The obtained polymerwas dissolved in 10 ml of tetrahydrofuran and dialyzed for 1 day with adialysis film (manufactured by Spectrum Inc., Stectra/Por StandardRegenerated Cellulose Dialysis Membrane 3), in a 1-L beaker containing500 ml of methanol. The polymer present in the dialysis film wasrecovered and dried under a reduced pressure to obtain 967 mg of adesired PHA.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=51000 and aweight-averaged molecular weight Mw=108000.

For specifying the structure of the obtained PHA, a NMR analysis wascarried out under conditions same as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenoxyvaleric acid representedby the following chemical formula (53), 3-hydroxy-5-cyclohexylvalericacid represented by the following chemical formula (68),3-hydroxy-9-carboxynonanoic acid represented by a chemical formula (54),3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula (55)and 3-hydroxy-5-carboxyvaleric acid represented by a chemical formula(56).

Also a proportion of the units of the obtained PHA was calculated by amethylesterification, utilizing trimethylsilyldiazomethane, of acarboxyl group at an end of a side chain of the PHA.

30 mg of the object PHA were charged in a 100-ml eggplant-shaped flaskand were dissolved by adding 2.1 ml of chloroform and 0.7 ml ofmethanol. The solution was added with 0.3 ml of a 2.0 mol/L solution oftrimethylsilyldiazomethane in hexane (supplied by Aldrich Inc.) and wasagitated for 30 minutes at the room temperature. After the reaction, thesolvent was distilled off in an evaporator to recover a polymer. Thepolymer was recovered by washing with 50 ml of methanol. A drying undera reduced pressure provided 28 mg of PHA.

A NMR analysis was carried out as in Example 1. As a result, ¹H-NMRspectrum confirmed a proportion of the units in which3-hydroxy-5-phenoxyvaleric acid was present by 49 mol %,3-hydroxy-5-cyclohexylvaleric acid by 42 mol %, a sum of three units of3-hydroxy-9-carboxynonanoic acid, 3-hydroxy-7-carboxyheptanoic acid and3-hydroxy-5-carboxyvaleric acid by 6 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 3 mol %.

Example 22

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemical Co.), 4 mmol/L of5-phenylvaleric acid, and 1 mmol/L of dodecanedioic acid monoethyl esterwere dissolved in 1000 ml of an aforementioned M9 culture medium, whichwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 5 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours at 30° C., wasadded to each prepared culture medium, and culture was conducted for 41hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 910 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=78000 and aweight-averaged molecular weight Mw=157000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60) by 78 mol %, three units of3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the followingchemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 14 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 8 mol %.

Example 23

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofyeast extract (supplied by DIFCO), 4 mmol/L of 5-phenylvaleric acid, and1 mmol/L of dodecanedioic acid monoethyl ester were dissolved in 1000 mlof an aforementioned M9 culture medium, which was placed in a 2000 mlshake flask, then sterilized in an autoclave and cooled to the roomtemperature. Then 5 ml of a culture liquid of Pseudomonas cichorii YN2strain, shake cultured in advance in an M9 culture medium containing0.5% of polypeptone for 8 hours at 30° C., was added to each preparedculture medium, and culture was conducted for 40 hours at 30° C. Afterthe culture, the cells were recovered by centrifuging, rinsed withmethanol and lyophilized. The dried cells, after weighing, were agitatedwith chloroform for 48 hours at 50° C. to extract a polymer. Thechloroform extract was filtered, then concentrated in an evaporator, anda solid precipitate formed with cold methanol was collected and driedunder a reduced pressure to obtain a desired polymer. According to aweighing of the obtained polymer, 250 mg (dry weight) of PHA wereobtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=75000 and aweight-averaged molecular weight Mw=152000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60) by 75 mol %, three units of3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the followingchemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 15 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 10 mol %.

Example 24

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofD-glucose (supplied by Kishida Kagaku), 4 mmol/L of 5-phenylvalericacid, and 1 mmol/L of dodecanedioic acid monoethyl ester were dissolvedin 1000 ml of an aforementioned M9 culture medium, which was placed in a2000 ml shake flask, then sterilized in an autoclave and cooled to theroom temperature. Then 5 ml of a culture liquid of Pseudomonas jesseniiP161 strain, shake cultured in advance in an M9 culture mediumcontaining 0.5% of polypeptone for 8 hours at 30° C., was added to eachprepared culture medium, and culture was conducted for 40 hours at 30°C. After the culture, the cells were recovered by centrifuging, rinsedwith methanol and lyophilized. The dried cells, after weighing, wereagitated with chloroform for 48 hours at 50° C. to extract a polymer.The chloroform extract was filtered, then concentrated in an evaporator,and a solid precipitate formed with cold methanol was collected anddried under a reduced pressure to obtain a desired polymer. According toa weighing of the obtained polymer, 300 mg (dry weight) of PHA wereobtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=71000 and aweight-averaged molecular weight Mw=149000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60) by 78 mol %, three units of3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the followingchemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 14 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 8 mol %.

Example 25

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemicals Co.), 4 mmol/L of5-phenoxyvaleric acid, and 1 mmol/L of dodecanedioic acid monoethylester were dissolved in 1000 ml of an aforementioned M9 culture medium,which was placed in a 2000 ml shake flask, then sterilized in anautoclave and cooled to the room temperature. Then 5 ml of a cultureliquid of Pseudomonas cichorii YN2 strain, shake cultured in advance inan M9 culture medium containing 0.5% of polypeptone for 8 hours at 30°C., was added to each prepared culture medium, and culture was conductedfor 41 hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 680 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=69000 and aweight-averaged molecular weight Mw=135000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenoxyvaleric acid representedby the following chemical formula (53) by 74 mol %, three units of3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the followingchemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 17 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 9 mol %.

Example 26

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemicals Co.), 4 mmol/L of4-cyclohexylbutyric acid, and 1 mmol/L of dodecanedioic acid monoethylester were dissolved in 1000 ml of an aforementioned M9 culture medium,which was placed in a 2000 ml shake flask, then sterilized in anautoclave and cooled to the room temperature. Then 5 ml of a cultureliquid of Pseudomonas cichorii YN2 strain, shake cultured in advance inan M9 culture medium containing 0.5% of polypeptone for 8 hours at 30°C., was added to each prepared culture medium, and culture was conductedfor 41 hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 720 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=81000 and aweight-averaged molecular weight Mw=160000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-4-cyclohexylbutyric acidrepresented by the following chemical formula (57) by 76 mol %, threeunits of 3-hydroxy-11-ethoxycarbonylundecanoic acid represented by thefollowing chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 16 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic-acid with 10 or 12 carbon atoms) by 8 mol %.

Example 27

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemicals Co.), 4 mmol/L of5-(phenylsulfanyl)valeric acid, and 1 mmol/L of dodecanedioic acidmonoethyl ester were dissolved in 1000 ml of an aforementioned M9culture medium, which was placed in a 2000 ml shake flask, thensterilized in an autoclave and cooled to the room temperature. Then 5 mlof a culture liquid of Pseudomonas cichorii YN2 strain, shake culturedin advance in an M9 culture medium containing 0.5% of polypeptone for 8hours at 30° C., was added to each prepared culture medium, and culturewas conducted for 42 hours at 30° C. After the culture, the cells wererecovered by centrifuging, rinsed with methanol and lyophilized. Thedried cells, after weighing, were agitated with chloroform for 48 hoursat 50° C. to extract a polymer. The chloroform extract was filtered,then concentrated in an evaporator, and a solid precipitate formed withcold methanol was collected and dried under a reduced pressure to obtaina desired polymer. According to a weighing of the obtained polymer, 890mg (dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=84000 and aweight-averaged molecular weight Mw=169000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(phenylsulfanyl)valeric acidrepresented by the following chemical formula (58) by 80 mol %, threeunits of 3-hydroxy-11-ethoxycarbonylundecanoic acid represented by thefollowing chemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 14 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 6 mol %.

Example 28

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemicals Co.), 4 mmol/L of5-benzoylvaleric acid, and 1 mmol/L of dodecanedioic acid monoethylester were dissolved in 1000 ml of an aforementioned M9 culture medium,which was placed in a 2000 ml shake flask, then sterilized in anautoclave and cooled to the room temperature. Then 5 ml of a cultureliquid of Pseudomonas cichorii YN2 strain, shake cultured in advance inan M9 culture medium containing 0.5% of polypeptone for 8 hours at 30°C., was added to each prepared culture medium, and culture was conductedfor 41 hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 450 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=156000 and aweight-averaged molecular weight Mw=325000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-benzoylvaleric acid representedby the following chemical formula (62) by 69 mol %, three units of3-hydroxy-11-ethoxycarbonylundecanoic acid represented by the followingchemical formula (69), 3-hydroxy-9-ethoxycarbonylnonanoic acidrepresented by a chemical formula (70), and3-hydroxy-7-ethoxycarbonylheptanoic acid represented by a chemicalformula (71) collectively by 18 mol %, and others (linear3-hydroxyalkanoic acid of 4 to 12 carbon atoms and 3-hydroxyalk-5-enoicacid with 10 or 12 carbon atoms) by 13 mol %.

Example 29

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemicals Co.), 4 mmol/L of5-(4-cyanophenoxy)valeric acid, and 1 mmol/L of sebacic acid monomethylester were dissolved in 1000 ml of an aforementioned M9 culture medium,which was placed in a 2000 ml shake flask, then sterilized in anautoclave and cooled to the room temperature. Then 5 ml of a cultureliquid of Pseudomonas cichorii YN2 strain, shake cultured in advance inan M9 culture medium containing 0.5% of polypeptone for 8 hours at 30°C., was added to each prepared culture medium, and culture was conductedfor 41 hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 450 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=68000 and aweight-averaged molecular weight Mw=129000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(4-cyanophenoxy)valeric acidrepresented by the following chemical formula (72) by 34 mol %, twounits of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by thefollowing chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoicacid represented by a chemical formula (74) collectively by 16 mol %,and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 50 mol %.

Example 30

There were prepared two 2000-ml shake flasks, and, in each, 0.1 wt. % ofn-nonanoic acid (supplied by Kishida Kagaku), 4 mmol/L of5-(4-nitrophenyl)valeric acid, and 1 mmol/L of sebacic acid monomethylester were dissolved in 1000 ml of an aforementioned M9 culture medium,which was placed in a 2000 ml shake flask, then sterilized in anautoclave and cooled to the room temperature. Then 5 ml of a cultureliquid of Pseudomonas cichorii YN2 strain, shake cultured in advance inan M9 culture medium containing 0.5% of polypeptone for 8 hours at 30°C., was added to each prepared culture medium, and culture was conductedfor 72 hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 170 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=59000 and aweight-averaged molecular weight Mw=125000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-(4-nitrophenyl)valeric acidrepresented by the following chemical formula (75) by 8 mol %, two unitsof 3-hydroxy-9-methoxycarbonylnonanoic acid represented by the followingchemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acidrepresented by a chemical formula (74) collectively by 18 mol %, andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 74 mol %.

Example 31

There were prepared two 2000-ml shake flasks, and, in each, 0.1 wt. % ofn-nonanoic acid (supplied by Kishida Kagaku), 4 mmol/L of5-[(phenylmethyl)oxy]valeric acid, and 1 mmol/L of sebacic acidmonomethyl ester were dissolved in 10000 ml of an aforementioned M9culture medium, which was placed in a 2000 ml shake flask, thensterilized in an autoclave and cooled to the room temperature. Then 5 mlof a culture liquid of Pseudomonas cichorii YN2 strain, shake culturedin advance in an M9 culture medium containing 0.5% of polypeptone for 8hours at 30° C., was added to each prepared culture medium, and culturewas conducted for 40 hours at 30° C. After the culture, the cells wererecovered by centrifuging, rinsed with methanol and lyophilized. Thedried cells, after weighing, were agitated with chloroform for 48 hoursat 50° C. to extract a polymer. The chloroform extract was filtered,then concentrated in an evaporator, and a solid precipitate formed withcold methanol was collected and dried under a reduced pressure to obtaina desired polymer. According to a weighing of the obtained polymer, 330mg (dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=79000 and aweight-averaged molecular weight Mw=152000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-[(phenylmethyl)oxy]valeric acidrepresented by the following chemical formula (68) by 81 mol %, twounits of 3-hydroxy-9-methoxycarbonylnonanoic acid represented by thefollowing chemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoicacid represented by a chemical formula (74) collectively by 13 mol %,and others (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 6 mol %.

Example 32

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemical Co.), 4 mmol/L of5-5-phenylvaleric acid, and 1 mmol/L of sebacic acid monomethyl esterwere dissolved in 1000 ml of an aforementioned M9 culture medium, whichwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 5 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours at 30° C., wasadded to each prepared culture medium, and culture was conducted for 40hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 1340mg (dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=81000 and aweight-averaged molecular weight Mw=159000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60) by 77 mol %, two units of3-hydroxy-9-methoxycarbonylnonanoic acid represented by the followingchemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acidrepresented by a chemical formula (74) collectively by 19 mol %, andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 4 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

The synthesized polyhydroxy alkanoate was formed into a film, and 500 mgof such film was placed on a Petri dish and was let to stand for 5 hoursin 100 ml of a 0.1N aqueous solution of sodium hydroxide. After thereaction, the sodium hydroxide solution was removed, and the polymer waswashed three times with 100 ml of distilled water. Then the polymer wasdissolved in 200 ml of ethyl acetate, and, after an addition of 100 mlof a 1.0N aqueous solution of hydrochloric acid, the solution wasagitated for 1 hour at the room temperature. Then the polymer wasextracted, washed with distilled water and the solvent was distilled offto recover the polymer. Thereafter, a drying under a reduced pressurewas carried out to obtain 350 mg of a desired polymer.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=9500 and aweight-averaged molecular weight Mw=32000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenylvaleric acid representedby the following chemical formula (60), 3-hydroxy-9-carboxynonanoic acidrepresented by the following chemical formula (54) and3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula(55).

Also a proportion of the units in the obtained polymer was calculatedfrom a decrease in ester groups, and confirmed as3-hydroxy-5-phenylvaleric acid by 78 mol %, 3-hydroxy-9-carboxylnonanoicacid and 3-hydroxy-7-carboxylheptanoic acid collectively by 12 mol %,3-hydroxy-9-methoxycarbonylnonanoic acid and3-hydroxy-7-methoxycarbonylheptanoic acid collectively by 6 mol %, andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 4 mol %.

Example 33

There were prepared two 2000-ml shake flasks, and, in each, 0.5 wt. % ofpolypeptone (supplied by Wako Pure Chemical Co.), 4 mmol/L of5-phenoxyvaleric acid, and 1 mmol/L of sebacic acid monomethyl esterwere dissolved in 1000 ml of an aforementioned M9 culture medium, whichwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 5 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours at 30° C., wasadded to each prepared culture medium, and culture was conducted for 40hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer. According to a weighing of the obtained polymer, 710 mg(dry weight) of PHA were obtained in the present example.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=71000 and aweight-averaged molecular weight Mw=148000.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenoxyvaleric acid representedby the following chemical formula (53) by 74 mol %, two units of3-hydroxy-9-methoxycarbonylnonanoic acid represented by the followingchemical formula (73) and 3-hydroxy-7-methoxycarbonylheptanoic acidrepresented by a chemical formula (74) collectively by 18 mol %, andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 8 mol %.

The polyhydroxy alkanoate thus obtained was utilized in the followingreaction.

The synthesized polyhydroxy alkanoate was formed into a film, and 500 mgof such film was placed on a Petri dish and was let to stand for 5 hoursin 100 ml of a 0.1N aqueous solution of sodium hydroxide. After thereaction, the sodium hydroxide solution was removed, and the polymer waswashed three times with 100 ml of distilled water. Then the polymer wasdissolved in 200 ml of ethyl acetate, and, after an addition of 100 mlof a 1.0N aqueous solution of hydrochloric acid, the solution wasagitated for 1 hour at the room temperature. Then the polymer wasextracted, washed with distilled water and the solvent was distilled offto recover the polymer. Thereafter, a drying under a reduced pressurewas carried out to obtain 370 mg of a desired polymer.

An average molecular weight of the obtained PHA was measured by gelpermeation chromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GELSuper HM-H, solvent: chloroform, converted to polystyrene). As a resultthere were obtained a number-averaged molecular weight Mn=8700 and aweight-averaged molecular weight Mw=30900.

For specifying the structure of the obtained PHA, a NMR analysis wasconducted under same conditions as in Example 1.

As a result, there was confirmed a polyhydroxy alkanoate copolymerincluding, as monomer units, 3-hydroxy-5-phenoxyvaleric acid representedby the following chemical formula (53), 3-hydroxy-9-carboxynonanoic acidrepresented by the following chemical formula (54) and3-hydroxy-7-carboxyheptanoic acid represented by a chemical formula(55).

Also a proportion of the units in the obtained polymer was calculatedfrom a decrease in ester groups, and confirmed as3-hydroxy-5-phenoxyvaleric acid by 73 mol %, 3-hydroxy-9-carboxynbnanoicacid and 3-hydroxy-7-carboxyheptanoic acid collectively by 10 mol %,3-hydroxy-9-methoxycarbonylnonanoic acid and3-hydroxy-7-methoxycarbonylheptanoic acid collectively by 8 mol %, andothers (linear 3-hydroxyalkanoic acid of 4 to 12 carbon atoms and3-hydroxyalk-5-enoic acid with 10 or 12 carbon atoms) by 9 mol %.

Example 34

In 1000 mL of an aforementioned M9 culture medium, there were added 0.5wt. % of polypeptone (supplied by Wako Pure Chemical Co.), and5-phenylvaleric acid and sebacic acid monomethyl ester so as to obtainfinal concentrations of 4 and 1 mmol/L respectively, and the solutionwas placed in a 2000 ml shake flask, then sterilized in an autoclave andcooled to the room temperature. Then 5 ml of a culture liquid ofPseudomonas cichorii YN2 strain, shake cultured in advance in an M9culture medium containing 0.5% of polypeptone for 8 hours at 30° C., wasadded to each prepared culture medium, and culture was conducted for 40hours at 30° C. After the culture, the cells were recovered bycentrifuging, rinsed with methanol and lyophilized. The dried cells,after weighing, were agitated with chloroform for 48 hours at 50° C. toextract a polymer. The chloroform extract was filtered, thenconcentrated in an evaporator, and a solid precipitate formed with coldmethanol was collected and dried under a reduced pressure to obtain adesired polymer.

A structure determination of the obtained polymer was carried out by amethynolysis-GC/MS method to be explained in the following. 5 mg of thepolymer were dissolved in 2 mL of chloroform, then added with 2 mL of a3% methanol solution of sulfuric acid and refluxed for 3.5 hours at 100°C. After the reaction, the reaction mixture was cooled to the roomtemperature and separated by adding 10 mL of deionized water underagitation. Then an organic layer was dehydrated with magnesium sulfate(anhydrous) and the reaction liquid was subjected to a measurement by agas chromatography-mass spectrometer (GC/MS: Shimadzu QP-5050A, column:DB-WAXETR 0.32 mm×30 m). An obtained total ion chromatogram (TIC) isshown in FIG. 5. There were observed three main peaks at 35.6, 38.0 and45.8 minutes. A mass spectrum (MS) of the peak at 35.6 minutes is shownin FIG. 6; a MS of the peak at 38.0 minutes is shown in FIG. 7; and a MSof the peak at 45.8 minutes is shown in FIG. 8.

As a result, the peak at 35.6 minutes was derived from a unit shown in achemical formula (80):

the peak at 38.0 minutes was derived from a unit shown in a chemicalformula (81):

and the peak at 35.6 minutes was derived from a unit shown in a chemicalformula (82):

Also proportions of the units, calculated from ratios of the peak areasof TIC, were 12.0%, 77.7% and 6.7% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography (GPC: Toso HLC-8220 GPC, column: Toso TSK-GEL Super HM-H,solvent: chloroform, converted to polystyrene). Table 1 shows weights ofthe obtained cells and the obtained polymer, a polymer weight ratio percell, The molecular weight and The molecular weight distribution of theobtained polymer.

TABLE 1 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 1358 67149.4 8.1 15.9 2.0 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight, Mw/Mn: molecular weightdistribution.

Example 35

A desired polymer was obtained in the same manner as in Example 34,except that the YN2 strain employed in Example 34 was replaced byPseudomonas jessenii P161 strain.

A structure determination of the obtained polymer carried out by amethanolysis-GC/MS method as in Example 34 confirmed that the polymerwas a polyhydroxy alkanoate copolymer constituted of units representedby chemical formulas (80), (81) and (82):

chemical formula (80):

chemical formula (81):

chemical formula (82):

and the proportions of the units, calculated from the ratios of the peakareas of TIC, were 15.5%, 75.2% and 9.3% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography as in Example 34.

Table 2 shows weights of the obtained cells and the obtained polymer, apolymer weight ratio per cell, the molecular weight and the molecularweight distribution of the obtained polymer.

TABLE 2 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 821 27133.0 6.6 13.9 2.1 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight, Mw/Mn: molecular weightdistribution.

Example 36

A desired polymer was obtained in the same manner as in Example 34,except that the YN2 strain employed in Example 34 was replaced byPseudomonas cichorii H45 strain and polypeptone was replaced by yeastextract (DIFCO).

A structure determination of the obtained polymer carried out by amethanolysis-GC/MS method as in Example 34 confirmed that the polymerwas a polyhydroxy alkanoate copolymer constituted of units representedby chemical formulas (80), (81) and (82):

chemical formula (80):

chemical formula (81):

chemical formula (82):

and the proportions of the units calculated from the ratios of the peakareas of TIC were 16.1%, 72.3% and 11.6% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography as in Example 34.

Table 3 shows weights of the obtained cells and the obtained polymer,polymer weight ratio per cell, the molecular weight and the molecularweight distribution of the obtained polymer.

TABLE 3 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 779 23029.5 7.2 14.9 2.1 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight, Mw/Mn: molecular weightdistribution.

Example 37

A desired polymer was obtained in the same manner as in Example 34,except that the YN2 strain employed in Example 34 was replaced byPseudomonas putida P91 strain and polypeptone was replaced by n-nonanoicacid (Kishida Kagaku, concentration: 0.1%).

A structure determination of the obtained polymer carried out by amethanolysis-GC/MS method as in Example 34 confirmed that the polymerwas a polyhydroxy alkanoate copolymer constituted of units representedby chemical formulas (80), (81), (82), (83), (84) and (85):

chemical formula (80)

chemical formula (81):

chemical formula (82)

chemical formula (83):

chemical formula (84):

chemical formula (85):

and the proportions of the units calculated from the ratios of the peakareas of TIC were 5.1%, 52.1%, 6.6%, 11.3%, 4.9% and 20.0% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography as in Example 34.

Table 4 shows weights of the obtained cells and the obtained polymer, apolymer weight ratio per cell, a molecular weight and a molecular weightdistribution of the obtained polymer.

TABLE 4 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 528 11020.8 8.2 16.9 2.1 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight, Mw/Mn: molecular weightdistribution.

Example 38

A desired polymer was obtained in the same manner as in Example 34,except that polypeptone was replaced by D-glucose (Kishida Kagaku).

A structure determination of the obtained polymer carried out by amethanolysis-GC/MS method as in Example 34 confirmed that the polymerwas a polyhydroxy alkanoate copolymer constituted of units representedby chemical formulas (80), (81) and (82):

chemical formula (80):

chemical formula (81):

chemical formula (82):

and the proportions of the units calculated from the ratios of the peakareas of TIC were 13.1%, 80.3% and 6.6% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography as in Example 34.

Table 5 shows weights of the obtained cells and the obtained polymer, apolymer weight ratio per cell, a molecular weight and a molecular weightdistribution of the obtained polymer.

TABLE 5 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 910 42546.7 7.9 15.4 1.9 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight, Mw/Mn: molecular weightdistribution.

Example 39

A desired polymer was obtained in the same manner as in Example 34,except that polypeptone was replaced by sodium pyruvate (KishidaKagaku).

A structure determination of the obtained polymer carried out by amethanolysis-GC/MS method as in Example 34 confirmed that the polymerwas a polyhydroxy alkanoate copolymer constituted of units representedby chemical formulas (80), (81) and (82):

chemical formula (80):

chemical formula (81):

chemical formula (82):

and the proportions of the units calculated from the ratios of the peakareas of TIC were 11.9%, 82.2% and 5.9% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography as in Example 34.

Table 6 shows weights of the obtained cells and the obtained polymer, apolymer weight ratio per cell, a molecular weight and a molecular weightdistribution of the obtained polymer.

TABLE 6 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 1120 58552.2 8.0 15.9 2.0 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight,Mw/Mn: molecular weight distribution.

Example 40

A desired polymer was obtained in the same manner as in Example 34,except that polypeptone employed in Example 34 was replaced by sodiumL-glutamate (Kishida Kagaku) and sebacic acid monomethyl ester, which isone of substrates for polymer synthesis was replaced by suberic acidmonomethyl ester.

A structure determination of the obtained polymer carried out by amethanolysis-GC/MS method as in Example 34 confirmed that the polymerwas a polyhydroxy alkanoate copolymer constituted of units representedby chemical formulas (86), (80) and (81):

chemical formula (86):

chemical formula (80):

chemical formula (81):

and the proportions of the units calculated from the ratios of the peakareas of TIC were 8.2%, 84.2% and 8.6% respectively.

The molecular weight of the polymer was measured by gel permeationchromatography as in Example 34.

Table 7 shows weights of the obtained cells and the obtained polymer, apolymer weight ratio per cell, a molecular weight and a molecular weightdistribution of the obtained polymer.

TABLE 7 Mw/ CDW (mg/L) PDW (mg/L) P/C % Mn (×10⁴) Mw (×10⁴) Mn 985 44044.7 7.8 14.6 1.9 CDW: cell dry weight, PDW: polymer dry weight, P/C:cell dry weight/polymer dry weight, Mn: number-averaged molecularweight, Mw: weight-averaged molecular weight, Mw/Mn: molecular weightdistribution.

1. A polyhydroxy alkanoate copolymer comprising at least a3-hydroxy-ω-alkenoic acid unit represented by a chemical formula (1) ina molecule, and simultaneously at least a 3-hydroxy-ω-alkanoic acid unitrepresented by a chemical formula (2) or a3-hydroxy-ω-cyclohexylalkanoic acid unit represented by a chemicalformula (3) in the molecule: chemical formula (1)

in which n represents an integer selected within a range indicated inthe chemical formula; and in case plural units are present, n is thesame or different for each unit; chemical formula (2)

in which m represents an integer selected within a range indicated inthe chemical formula; R represents a residue having any of a phenylstructure or a thienyl structure; and in case plural units are present,m and R are the same or different for each unit; chemical formula (3)

in which R₁ being a substituent on a cyclohexyl group represents ahydrogen atom, a CN group, a NO₂ group, a halogen atom, a CH₃ group, aC₂H₅ group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F₇ group; krepresents an integer selected within a range indicated in the chemicalformula; and in case plural units are present, R₁ and k may be the sameor different for each unit, wherein the polyhydroxy alkanoate copolymeris biosynthesized by using a microorganism capable of producing it withat least an ω-alkenoic acid represented by a chemical formula (24) andat least a compound represented by a chemical formula (25) or at leastan ω-cyclohexylalkanoic acid represented by a chemical formula (26) asstarting materials: chemical formula (24)

in which p represents an integer selected within a range indicated inthe chemical formula: chemical formula (25)

in which q represents an integer selected within a range indicated inthe chemical formula: and R₂₃ is a residue having a phenyl structure ora thienyl structure; and chemical formula (26)

in which R₂₄ is a substituent on a cyclohexyl group and represents an Hatom, a CN group, a NO₂ group, a halogen atom, a CH₃ group, a C₂H₅group, a C₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F₇ group: and rrepresents an integer selected within a range indicated in the chemicalformula.
 2. The polyhydroxy alkanoate copolymer according to claim 1,wherein the residue having a phenyl structure or a thienyl structure ofR in the chemical formula (2) and of R₂₃ in the chemical formula (25) isselected from the group consisting of chemical formulas (8), (9), (10),(11), (12), (13), (14), (15), (16), (17) and (18): the chemical formula(8):

represents a group of unsubstituted or substituted phenyl groups inwhich R₂ is a substituent on an aromatic ring and represents an H atom,a halogen atom, a CN group, a NO₂ group, a CH₃ group, a C₂H₅ group, aC₃H₇ group, a CH═CH₂ group, a COOR₃ group (R₃ represents an H atom, a Naatom or a K atom), a CF₃ group, a C₂F₅ group, or a C₃F₇ group; and incase plural units are present, R₂ is the same or different for eachunit; the chemical formula (9):

represents a group of unsubstituted or substituted phenoxy groups inwhich R₄ is a substituent on an aromatic ring and represents an H atom,a halogen atom, a CN group, a NO₂ group, a CH₃ group, a C₂H₅ group, aC₃H₇ group, a SCH₃ group, a CF3 group, a C₂F₅ group, or a C₃F₇ group;and in case plural units are present, R₄ may be the same or differentfor each unit; the chemical formula (10):

represents a group of unsubstituted or substituted benzoyl groups inwhich R₅ is a substituent on an aromatic ring and represents an H atom,a halogen atom, a CN group, a NO₂ group, a CH₃ group, a C₂H₅ group, aC₃H₇ group, a CF₃ group, a C₂F₅ group, or a C₃F7 group; and in caseplural units are present, R₅ may be the same or different for each unit;the chemical formula (11)

represents a group of substituted or unsubstituted phenylsulfanyl groupsin which R₆ is a substituent on an aromatic ring and represents an Hatom, a halogen atom, a CN group, a NO₂ group, a COOR₇ group, a SO₂R₈group (R₇ represents either one of H, Na, K, CH₃ and C₂H₅; and R₈represents either one of OH, ONa, OK, a halogen atom, OCH₃ and OC₂H₅), aCH₃ group, a C₂H₅ group, a C₃H₇ group, a (CH₃)₂—CH group or a (CH₃)₃—Cgroup; and in case plural units are present, R₆ may be the same ordifferent for each unit; the chemical formula (12):

represents a group of substituted or unsubstituted(phenylmethyl)sulfanyl groups in which R₉ is a substituent on anaromatic ring and represents an H atom, a halogen atom, a CN group, aNO₂ group, a COOR₁₀ group, a SO₂R₁₁ group (R₁₀ represents either one ofH, Na, K, CH₃ and C₂H₅; and R₁₁ represents either one of OH, ONa, OK, ahalogen atom, OCH₃ and OC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group,a (CH₃)₂—CH group or a (CH₃)₃—C group; and in case plural units arepresent, R₉ may be the same or different for each unit; the chemicalformula (13):

represents a 2-thienyl group; the chemical formula (14)

represents a 2-thienylsulfanyl group; the chemical formula (15):

represents a 2-thienylcarbonyl group; the chemical formula (16):

represents a group of substituted or non-substituted phenylsulfinylgroups in which R₁₂ is a substituent on an aromatic ring and representsan H atom, a halogen atom, a CN group, a NO₂ group, a COOR₁₃ group, aSO₂R₁₄ group (R₁₃ represents either one of H, Na, K, CH₃ and C₂H₅; andR₁₄ represents either one of OH, ONa, OK, a halogen atom, OCH₃ andOC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group, a (CH₃)₂—CH group or a(CH₃)₃—C group; and in case plural units are present, R₁₂ may be thesame or different for each unit; the chemical formula (17):

represents a group of substituted or non-substituted phenylsulfonylgroups in which R₁₅ is a substituent on an aromatic ring and representsan H atom, a halogen atom, a CN group, a NO₂ group, a COOR₁₆ group, aSO₂R₁₇ group (R₁₆ represents either one of H, Na, K, CH₃ and C₂H₅; andR₁₇ represents either one of OH, ONa, OK, a halogen atom, OCH₃ andOC₂H₅), a CH₃ group, a C₂H₅ group, a C₃H₇ group, a (CH₃)₂—CH group or a(CH₃)₃—C group; and in case plural units are present, R₁₅ may be thesame or different for each unit; and the chemical formula (18):

represents a (phenylmethyl)oxy group.
 3. The polyhydroxy alkanoatecopolymer according to claim 1, which has a number-averaged molecularweight within a range from 1000 to 1000000.